Minimized cyanobacterial microcompartment for carbon dioxide fixation

ABSTRACT

A fusion chimeric protein is described herein that can assemble a functional carboxysome core, which is able to fix carbon by taking atmospheric carbon dioxide and converting it into useful carbon-containing compounds such as 3-phosphoglycerate (3-PGA).

This application claims benefit of priority to the filing date of U.S. Provisional Application Ser. No. 62/378,979, filed Aug. 24, 2016, the contents of which are specifically incorporated herein by reference in their entity.

FEDERAL FUNDING

This invention was made with government support under DE-FG02-91ER20021 awarded by the U.S. Department of Energy. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Like plants and algae, cyanobacteria obtain energy from photosynthesis, utilizing energy from sunlight and electrons from water to reduce carbon dioxide (CO₂) and thereby ‘fix’ carbon into cell biomass. This photosynthetically-fixed carbon can then be used to make metabolites, such as carbohydrates, proteins, and fatty acids that are ultimately distributed to heterotrophic organisms. Besides their role as primary carbon fixation organisms, cyanobacteria can also be altered to produce useful products. For example, Synechococcus elongatus PCC 7942 has been engineered to produce isobutyraldehyde and butanol; Synechocystis sp. PCC 6803 has been modified produce ethanol and isoprene.

Cyanobacteria excel at carbon fixation, thanks to their complex carbon concentrating mechanism (ccm), which is comprised of bicarbonate pumps, carbon dioxide-uptake systems and the carboxysome. The carboxysome is an approximate 300 MDa compartment essential for carbon concentration, as it enhances carbon fixation by sequestering ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase (CA) within a protein shell. In the carboxysome lumen, bicarbonate is converted into carbon dioxide by carbonic anhydrase. Such conversion increases the proportion of carbon dioxide to oxygen in the vicinity of Rubisco, which favors Rubisco's carboxylase activity, while the shell limits the loss of carbon dioxide into the bulk cytosol (Cai et. al, 2009).

Researchers have explored ways to express the β-carboxysome shell and cyanobacterial form 1B Rubisco in chloroplasts (Lin et al., 2014b; Lin et al., 2014a). However, constructs have not been generated that can assemble the functional multi-protein metabolic carboxysome core.

SUMMARY

In cyanobacteria, the key enzyme for photosynthetic CO₂ fixation, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is bound within proteinaceous polyhedral microcompartments called carboxysomes. A streamlined carboxysome is described herein that was generated by fusing key domains from four proteins into a single protein. This chimeric protein assembles into a functional carboxysome core that can readily be transferred and utilized in other organisms. This is the first instance of the redesign and construction of a carboxysome core, the first instance of a re-design of a bacterial microcompartment core, and lays the base for the generation of novel compartments with industrially relevant functions based on the carboxysome and related bacterial microcompartment architectures.

Described herein are fusion proteins that include a polypeptide comprising at least two small subunit-like domains (SSLDs) from a carbon dioxide concentrating mechanism (CcmM) protein, at least one carbonic anhydrase domain, and at least one encapsulation peptide. The at least two small subunit-like domains (SSLDs) from a carbon dioxide concentrating mechanism (CcmM) protein can bind or nucleate with ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The Rubisco can, for example, synthesize 3-phosphoglycerate (3-PGA). In some cases, the at least two small subunit-like domains (SSLDs) from a carbon dioxide concentrating mechanism (CcmM) protein can have a protein sequence with at least 95% sequence identity to any of SEQ ID NO:1-11, 37, 75, 76, or 77.

The at least one carbonic anhydrase domain is an enzyme that can convert bicarbonate to carbon dioxide. For example, the at least one carbonic anhydrase domain comprises at least 95% sequence identity to any of SEQ ID NO:17-21 or 71.

The at least one encapsulation peptide can interact with, nucleate, and/or bind one or more carboxysome shell protein. In some cases, the at least one encapsulation peptide comprises at least 95% sequence identity to any of SEQ ID NO:12-15 or 16.

Also described herein are expression cassettes that can include a promoter operably linked to a nucleic acid segment encoding such a fusion protein. Cells, plants, bacteria, algae, and/or microalgae can be modified to include such expression cassettes.

Methods are also described herein that can provide carbon fixation. Such methods can include culturing the cells that have nucleic acids or expression vectors that encode any of the fusion proteins described herein. The methods can involve cultivating one or more plants that have nucleic acids or expression vectors that encode any of the fusion proteins described herein. Such cells, plants, bacteria, algae, and/or microalgae can manufacture products such as 3-phosphoglycerate (3-PGA). Such cells, plants, bacteria, algae, and/or microalgae can be cultivated or cultured and then harvested. Products can be harvested from the cells, plants, bacteria, algae, and/or microalgae. Such products can include oils, carbohydrates, grains, vegetables, fruits and other components, as well as 3-phosphoglycerate (3-PGA).

DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1A-1H illustrate construction of a chimeric cyanobacterial carboxysome core. FIG. 1A is a schematic diagram of the architecture of domains in the CcmM, CcmN, CcaA and CcmC proteins that recruit Rubisco and assemble the carboxysome core. FIG. 1B schematically illustrates the architecture and structure of the domains of the constructed chimeric protein, CcmC. SSLD: Small subunit-like domain. EP: Encapsulation peptide. CA: Carbonic anhydrase. FIG. 1C is a schematic diagram of the native β-carboxysome core protein. The shell of the native β-carboxysome core protein includes the CcmL (pentagon), CcmO (hexagon), and CcmK (hexagon) polypeptides encoded by the ccm operon. The core of the native β-carboxysome core protein includes the other proteins encoded by the ccm operon. FIG. 1D is a schematic diagram of the CcaA-M35 construct, which is a fusion of CcaA and 3×SSLDs, and which was determined to not be a successful design. FIG. 1E is a schematic diagram of the M35-EP construct, which is a fusion of the Encapsulation peptide of CcmN and three SSLDs, and which was also determined to not be a successful design. FIG. 1F is a schematic diagram of the CcmC fusion of the Encapsulation peptide of CcmN, a shortened version of CcaA (central flattened circle linked to a pentagon), and three SSLDs (three rectangles at the bottom that bind or nucleate with the CcaA). The shell of the chimeric cyanobacterial carboxysome protein includes the CcmL (pentagon), CcmO (hexagon), and CcmK (hexagon) polypeptides encoded by the ccm operon. Gray shading between polypeptide domains denotes known non-covalent domain interactions. SSLD: small subunit-like domain. EP: Encapsulation peptide. FIG. 1G illustrates assembly of a native β-carboxysome core. FIG. 1H illustrates assembly of the designed carboxysome core by the chimeric protein CcmC (yellow). For FIGS. 1G and 1H, the small subunit-like domains (SSLDs) are numbered from the N-terminal (SSLD1) to C-terminal (SSLD3). The specific details of their interactions with the large subunit of Rubisco are unknown, but they may displace some of the RbcS subunits, which are not shown. Domains are colored as in FIGS. 1A and 1B. Shell proteins are shown in blue, while four RbcL subunits of the L8S8 complex of Rubisco are shown in green. Gray shading denotes known noncovalent domain interactions with a numbers in parenthesis for the corresponding reference: (1) and (2) from Kinney et al. (2012), (3) from Long et al. (2007) and (4) from Long et al. (2010).

FIG. 2 illustrates cross-reactivity of the chimeric protein CcmC with anti-CcmM antibodies. Whole cell lysates were blotted and probed using anti-CcmM antibodies. Protein extracts from Controls (Wild-type background) show two bands corresponding to the full length and the short form of CcmM, while the mutants (CcmC background) show one band due to the cross-reactivity of the antibody with the small subunit-like domains.

FIG. 3 illustrates structural complementation of the carboxysome core deletion strains with the chimeric protein CcmC. FIG. 3 panels A-D show fluorescence of cyanobacteria strains expressing RbcL-GFP for carboxysome visualization by microscopy. FIG. 3 panel A illustrates fluorescence of wild-type/RbcL-GFP cyanobacteria. FIG. 3 panel B illustrates fluorescence of COREΔ2/RbcL-GFP cyanobacteria. FIG. 3 panel C illustrates fluorescence of COREΔ2/CcmC/RbcL-GFP cyanobacteria. FIG. 3 panel D illustrates fluorescence of COREΔ3/CcmC/RbcL-GFP cyanobacteria. Scale bar: 5 μm. FIG. 3 panels E-H show electron micrographs of the same strains after incubation for at least 12 hours in air. FIG. 3 panel E shows images of wild-type/RbcL-GFP cyanobacteria. FIG. 3 panel F shows images of COREΔ2/RbcL-GFP cyanobacteria. FIG. 3 panel G shows images of COREΔ2/CcmC/RbcL-GFP cyanobacteria. FIG. 3 panel H shows images of COREΔ3/CcmC/RbcL-GFP cyanobacteria. Arrowheads: carboxysomes. Scale bar: 500 nm.

FIG. 4A-4C illustrates the structural features of native and minimized carboxysomes. FIG. 4A graphically illustrates the distribution of the number of carboxysomes per cell (n≤100), where the solid line (solid circle symbols) shows results for wild-type/RbcL-GFP, the dash-dotted line (triangle symbols) shows results for COREΔ2/CcmC/RbcL-GFP, and the dashed line (square symbols) shows results for COREΔ3/CcmC/RbcL-GFP. FIG. 4B graphically illustrates the relative RbcL content in protein samples normalized to Chlorophyll a (n=3), where the dark gray bar shows results for wild-type/RbcL-GFP, the open (white) bar shows results for COREΔ2/CcmC/RbcL-GFP, and the light gray bar shows results for COREΔ3/CcmC/RbcL-GFP. FIG. 4C graphically illustrates the carboxysome diameters measured from electron micrographs (n=50), where the dark gray bar shows results for wild-type/RbcL-GFP, the open (white) bar shows results for COREΔ2/CcmC/RbcL-GFP, and the light gray bar shows results for COREΔ3/CcmC/RbcL-GFP. Error bars=std. dev.

FIG. 5A-5B illustrate growth of CcmC strains compared to wild type showing that functional complementation has occurred of the carboxysome core deletion by the chimeric protein, CcmC. FIG. 5A shows the changes in optical density (730 nm) over time of independent cultures grown in air (n=3). Wild type/RbcL-GFP (circles), COREΔ2/CcmC/RbcL-GFP (triangles), and COREΔ3/CcmC/RbcL-GFP (squares) show similar growth rates. Note that the COREΔ2/RbcL-GFP (without the CcmC construct; diamond symbols) failed to grow in air. The inset chart shows doubling times calculated by exponential regression curve fitting (see website at doubling-time.com/compute.php). FIG. 5B shows the changes in optical density (730 nm) over time of independent cultures grown in 5% CO₂ (n=3). Wild type/RbcL-GFP (circles), COREΔ2/CcmC/RbcL-GFP (triangles), and COREΔ3/CcmC/RbcL-GFP (squares) show similar growth rates when detected by optical density (730 nm). Error bars=std. dev. The inset chart shows doubling times calculated by exponential regression curve fitting (see website at doubling-time.com/compute.php).

FIG. 6A-6C illustrate physiological parameters of wild-type vs. CcmC strains. FIG. 6A graphically illustrates the average absorbance spectra of whole cell suspensions normalized to Chlα (663 nm), where the solid line shows data for wild-type/RbcL-GFP cells, and the dashed line shows data for COREΔ3/CcmC/RbcL-GFP cells (n=3). FIG. 6B graphically illustrates changes over time of F_(v)/F_(m) in cultures grown at 3% CO₂ and transferred to air at time=0 h, where the solid line shows data for wild-type/RbcL-GFP cells (circle symbols), the dashed line shows data for COREΔ3/CcmC/RbcL-GFP cells (square symbols), and the dashed, dotted line shows data for COREΔ2/RbcL-GFP cells (triangle symbols) (n=3). FIG. 6C graphically illustrates oxygen evolution rates (normalized to Chl α) at high light intensity of strains grown in air and supplemented with 10 mM bicarbonate (left) with a comparison of Chl α per ml of OD₇₃₀ culture (right), where the dark grey bars are data for wild-type/RbcL-GFP cells and the light grey bars are data for COREΔ3/CcmC/RbcL-GFP cells. (n≤5). Error bars=std. dev.

DETAILED DESCRIPTION

A chimeric protein is described herein that can assemble into a functional carboxysome core and that is able to fix carbon by taking atmospheric carbon dioxide and converting it into useful carbon-containing molecules such as 3-phosphoglycerate (3-PGA or also referred to as glycerate 3-phosphate). 3PGA is a precursor for other useful molecules such as serine, which, in turn, can create cysteine and glycine through the homocysteine cycle.

The chimeric protein is referred to as CcmC (where the final “C” is for chimeric). The CmcC protein structure is schematically illustrated in FIGS. 1B and 1H. The chimeric CcmC protein can be expressed in a variety of organisms. For example, although the CcmC protein has been generated from cyanobacterial components, it can be expressed in a variety of organisms such as bacteria, plants, microalgae and other organisms to assemble organelles that remove carbon dioxide from the atmosphere and provide organic carbon to facilitate growth and synthesis of useful products. The chimeric protein does not exist in nature, it was designed and synthesized recombinantly.

The chimeric protein structurally and functionally replaces four gene products required for carboxysome formation (see schematic illustrations in FIGS. 1B and 1H). The CcmC protein contains scaffolding domains (the Small RbcS subunit-like domains that are involved in nucleating Ribulose-1,5-bisphosphate carboxylase/oxygenase (commonly known by the abbreviation Rubisco), an enzymatic domain (carbonic anhydrase), and an encapsulating domain (the encapsulation peptide).

Functional carboxysomes are needed for the survival of a cyanobacterial host. As illustrated herein, the chimeric CcmC protein can replace the function of native carboxysomes in cyanobacteria.

In CcmC, the small subunit-like domains (SSLDs) and the Encapsulation peptide (EP) are fused to opposite ends of the beta-carbonic anhydrase (β-CA) domain. The SSLDs are available to interact with the large subunit of Rubisco and the Encapsulation peptide can interact with the shell (see FIGS. 1H and 1B). The resulting 67 kDa chimeric protein replaces the 58 kDa CcmM, the 35 kDa M35, the 16 kDa CcmN, and the 30 kDa CcaA proteins that are part of the native (wild type) cyanobacterial carboxysome core protein.

The CcmC construct reduces the genomic load required to assemble a carboxysome by about 1100 bp, which is about 18% of total message required for wild type carboxysomes. In addition, it reduces the number of proteins and, concomitantly, the need to balance the expression levels of four different genes.

The chimeric CcmC carboxysomes, although smaller, morphologically resemble wild-type carboxysomes (FIG. 3) and they are able to support photosynthesis (FIG. 5A-5B). The results provided herein demonstrate that at least four protein domains can be combined into one, and that a non-native fusion protein can be enclosed in carboxysome shells by including a single encapsulating domain (the EP) as part of the chimeric CcmC protein.

Carboxysomes

Bacterial microcompartments (BMCs) are a family of architecturally similar but functionally diverse self-assembling organelles composed entirely of protein (Axen et al., 2014; Kerfeld and Erbilgin, 2015). The first BMC identified was the carboxysome (Drews and Niklowitz, 1956). Carboxysomes are about 300 MDa in size. Carboxysomes form compartments (Cheng et al., 2008) that are part of the cyanobacterial carbon concentrating mechanism (ccm) that enhance carbon fixation by sequestering ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase (CA) within a protein shell. In the carboxysome lumen, bicarbonate is converted into carbon dioxide by a carbonic anhydrase (CA), which increases the proportion of carbon dioxide to oxygen in the vicinity of Rubisco while the carboxysome shell limits the loss of carbon dioxide into the bulk cytosol (Cai et. al, 2009). Such increased concentration of carbon dioxide favors Rubisco's carboxylase activity. The product of carbon fixation, 3-phosphoglycerate (3-PGA), exits the carboxysome and can be used in the Calvin cycle or other biosynthetic pathways. Rubisco is the most abundant protein in the biosphere and is responsible for the majority of Earth's primary production of biomass.

Two types of carboxysomes are found in cyanobacteria: α-carboxysomes containing form 1A Rubisco, and β-carboxysomes containing form 1B Rubisco. The constituent core proteins also differ between the two types of carboxysomes, as well as the mode of assembly. Recently it was proposed that a large, conserved multi-domain protein (CsoS2) organizes the Rubisco in the α-carboxysome core (Cai et al., 2015). In contrast, assembly of the β-carboxysome involves a sequence of protein domain interactions among multiple core proteins (Cameron et al., 2013).

In Synechococcus elongatus PCC 7942, the β-carboxysome shell is formed by the structural proteins CcmK, CcmL and CcmO. The core of native carboxysomes is composed of CcmM, M35 and CcmN as well as the enzymes Rubisco (form 1B) and the β-carbonic anhydrase, CcaA (FIG. 1G).

CcmM Protein

The carbon dioxide concentrating mechanism protein, CcmM, can exist as 58-kDa and 35-kDa protein products in Synechococcus elongatus PCC 7942. The relative composition of the 58-kDa and 35-kDa CcmM proteins is not affected by protease inhibitors. FIG. 1A shows a schematic diagram of the CcmM protein.

An amino acid sequence for a Synechococcus elongatus PCC 7942 carbonate dehydratase (CcmM: Synpcc7942_1423; 57833 daltons) is available as accession number ABB57453 (see website at uniprot.org/uniprot/Q03513)(SEQ ID NO:1).

1 MPSPTTVPVA TAGRLAEPYI DPAAQVHAIA SIIGDVRIAA 41 GVRVAAGVSI RADEGAPFQV GKESILQEGA VIHGLEYGRV 81 LGDDQADYSV WIGQRVAITH KALIHGPAYL GDDCFVGFRS 121 TVFNARVGAG SVIMMHALVQ DVEIPPGRYV PSGAIITTQQ 161 QADRLPEVRP EDREFARHII GSPPVIVRST PAATADFHST 201 PTPSPLRPSS SEATTVSAYN GQGRLSSEVI TQVRSLLNQG 241 YRIGTEHADK RRFRTSSWQP CAPIQSTNER QVLSELENCL 281 SEHEGEYVRL LGIDTNTRSR VFEALIQRPD GSVPESLGSQ 321 PVAVASGGGR QSSYASVSGN LSAEVVNKVR NLLAQGYRIG 361 TEHADKRRFR TSSWQSCAPI QSSNERQVLA ELENCLSEHE 401 GEYVRLLGID TASRSRVFEA LIQDPQGPVG SAKAAAAPVS 441 SATPSSHSYT SNGSSSSDVA GQVRGLLAQG YRISAEVADK 481 RRFQTSSWQS LPALSGQSEA TVLPALESIL QEHKGKYVRL 521 IGIDPAARRR VAELLIQKP

A related CcmM protein from Synechococcus elongatus has a sequence has at least 99% sequence identity to SEQ ID NO:1, as illustrated below (SEQ ID NO:2).

99.8% identity in 539 residues overlap; Score: 2722.0; Gap frequency: 0.0% Seq1 1 MPSPTTVPVATAGRLAEPYIDPAAQVHAIASIIGDVRIAAGVRVAAGVSIRADEGAPFQV Seq2 1 MPSPTTVPVATAGRLAEPYIDPAAQVHAIASIIGDVRIAAGVRVAAGVSIRADEGAPFQV ************************************************************ Seq1 61 GKESILQEGAVIHGLEYGRVLGDDQADYSVWIGQRVAITHKALIHGPAYLGDDCFVGFRS Seq2 61 GKESILQEGAVIHGLEYGRVLGDDQADYSVWIGQRVAITHKALIHGPAYLGDDCFVGFRS ************************************************************ Seq1 121 TVFNARVGAGSVIMMHALVQDVEIPPGRYVPSGAIITTQQQADRLPEVRPEDREFARHII Seq2 121 TVFNARVGAGSVIMMHALVQDVEIPPGRYVPSGAIITTQQQADRLPEVRPEDREFARHII ************************************************************ Seq1 181 GSPPVIVRSTPAATADFHSTPTPSPLRPSSSEATTVSAYNGQGRLSSEVITQVRSLLNQG Seq2 181 GSPPVIVRSTPAATADFHSTPTPSPLRPSSSEATTVSAYNGQGRLSSEVITQVRSLLNQG ************************************************************ Seq1 241 YRIGTEHADKRRFRTSSWQPCAPIQSTNERQVLSELENCLSEHEGEYVRLLGIDTNTRSR Seq2 241 YRIGTEHADKRRFRTSSWQPCAPIQSTNERQVLSELENCLSEHEGEYVRLLGIDTNTRSR ************************************************************ Seq1 301 VFEALIQRPDGSVPESLGSQPVAVASGGGRQSSYASVSGNLSAEVVNKVRNLLAQGYRIG Seq2 301 VFEALIQRPDGSVPESLGSQPVAVASGGGRQSSYASVSGNLSAEVVNKVRNLLAQGYRIG ************************************************************ Seq1 361 TEHADKRRFRTSSWQSCAPIQSSNERQVLAELENCLSEHEGEYVRLLGIDTASRSRVFEA Seq2 361 TEHADKRRFRTSSWQSCAPIQSSNERQVLAELENCLSEHEGEYVRLLGIDTASRSRVFEA ************************************************************ Seq1 421 LIQDPQGPVGSAKAAAAPVSSATPSSHSYTSNGSSSSDVAGQVRGLLAQGYRISAEVADK Seq2 421 LIQDPQGPVGSAKAAAAPVSSATPSSHSYTSNGSSSSDVAGQVRGLLAQGYRISAEVADK ************************************************************ Seq1 481 RRFQTSSWQSLPALSGQSEATVLPALESILQEHKGKYVRLIGIDPAARRRVAELLIQKP Seq2 481 RRFQTSSWQSLPALSGRSEATVLPALESILQEHKGKYVRLIGIDPAARRRVAELLIQKP **************** ****************************************** This related protein has a sequence that is available from the National Center for Biotechnology Information database (see website at ncbi.nlm.nih.gov) with accession number WP_011242447.1 (GI:499561664), and with the sequence shown below (SEQ ID NO:2)

1 MPSPTTVPVA TAGRLAEPYI DPAAQVHAIA SIIGDVRIAA 41 GVRVAAGVSI RADEGAPFQV GKESILQEGA VIHGLEYGRV 81 LGDDQADYSV WIGQRVAITH KALIHGPAYL GDDCFVGFRS 121 TVFNARVGAG SVIMMHALVQ DVEIPPGRYV PSGAIITTQQ 161 QADRLPEVRP EDREFARHII GSPPVIVRST PAATADFHST 201 PTPSPLRPSS SEATTVSAYN GQGRLSSEVI TQVRSLLNQG 241 YRIGTEHADK RRFRTSSWQP CAPIQSTNER QVLSELENCL 281 SEHEGEYVRL LGIDTNTRSR VFEALIQRPD GSVPESLGSQ 321 PVAVASGGGR QSSYASVSGN LSAEVVNKVR NLLAQGYRIG 361 TEHADKRRFR TSSWQSCAPI QSSNERQVLA ELENCLSEHE 401 GEYVRLLGID TASRSRVFEA LIQDPQGPVG SAKAAAAPVS 441 SATPSSHSYT SNGSSSSDVA GQVRGLLAQG YRISAEVADK 481 RRFQTSSWQS LPALSGRSEA TVLPALESIL QEHKGKYVRL 521 IGIDPAARRR VAELLIQKP

A related CcmM protein from Prochlorothrix hollandica has a sequence has at least 53% sequence identity to SEQ ID NO:1, as illustrated below (SEQ ID NO:3).

51.5% identity in 563 residues overlap; Score: 1331.0; Gap frequency: 4.8% Seq1 3 SPTTVPVATAGRLAEPYIDPAAQVHAIASIIGDVRIAAGVRVAAGVSIRADEGAPFQVGK Seq3 5 SSAAPPTPWSRGLAEPQIDGSAYVHAFSNVIGDVWIGENVLIAPGTSIRADEGAPFHIGS *    *      **** **  * ***    **** *   *  * * **********  * Seq1 63 ESILQEGAVIHGLEYGRVLGDDQADYSVWIGQRVAITHKALIHGPAYLGDDCFVGFRSTV Seq3 65 STNIQDGVVIHGLEQGRVLGDDQKEYSVWVGRDSSLTHKALIHGPAYVGDECFIGFRSTV     * * ****** ********  **** *     *********** ** ** ****** Seq1 123 FNARVGAGSVIMMHALVQDVEIPPGRYVPSGAIITTQQQADRLPEVRPEDREFARHIIGS Seq3 125 FNARVGHGCIVMMHALIQDVEIPPGKYVPSGAIITSQQQADRLPDVRQEDKDFAHHVVGI ****** *   ***** ******** ********* ******** ** **  ** *  * Seq1 183 PPVIVRSTPAATADFHSTPTPSPLRPSSSEAT------------TVSAYNGQGRLSSEVI Seq3 185 NEALLAGYHCARSSACINPIRAGLSQTFQGSTPGTHGLEESINGTTNTMNNGYGLSPALI           *       *    *       *            *    *    **   * Seq1 231 TQVRSLLNQGYRIGTEHADKRRFRTSSWQPCAPIQSTNERQVLSELENCLSEHEGEYVRL Seq3 245 SQVRSLLAQGYRIGTEHATPRRFKTSSWESCAPIESKNEGQVLSALSGCLQEHQGEYVRL  ****** **********  *** ****  **** * ** **** *  ** ** ****** Seq1 291 LGIDTNTRSRVFEALIQRPDGSVPE--SLGSQPVAVASGGGRQSSYASVSGNLSAEVVNK Seq3 305 LGIDVQARRRVLEVLIQRPDGKPTSLSTRGTVSVAAPSASNGHRSSTAGTSNGGGSLADQ ****   * ** * *******        *   **  *      *      * Seq1 349 VRNLLAQGYRIGTEHADKRRFRTSSWQSCAPIQSSNERQVLAELENCLSEHEGEYVRLLG Seq3 365 VRGLLQQGCRITTEHADKRRFKTSSWQVGAVIESSNFNQVMAALDSAMQQYSGEYVRLIA ** ** ** ** ********* *****  * * ***  ** * *        ****** Seq1 409 IDTASRSRVFEALIQDPQGPVGSAKAAAAPVSSATPSSHSYTSNGSSSS----------- Seq3 425 VDPLAKRRVAEVLIHRPDGKPVATTAASKGSTYSSNGASNGASNGASSNGYGGGSVSGLS  *     ** * **  * *      **               *** ** Seq1 458 -DVAGQVRGLLAQGYRISAEVADKRRFQTSSWQSLPALSGQSEATVLPALESILQEHKGK Seq3 485 GETANQVRGWLGQGYRISAEYADKRRFKTGSWQTHGTLEGRGDQ-VLGSISTVLSTHSGN    * **** * ******** ****** * ***    * *     **      *  * * Seq1 517 YVRLIGIDPAARRRVAELLIQKP Seq3 544 YVRLVGVDPQAKRRVGQVIIQRP **** * ** * ***    ** * This related CcmM protein from Prochlorothrix hollandica has a sequence that is available from the National Center for Biotechnology Information database (see website at ncbi.nlm.nih.gov) with accession number WP_017713783.1 (GI:516317089), and with the sequence shown below (SEQ ID NO:3).

1 MAGYSSAAPP TPWSRGLAEP QIDGSAYVHA FSNVIGDVWI 41 GENVLIAPGT SIRADEGAPF HIGSSTNIQD GVVIHGLEQG 81 RVLGDDQKEY SVWVGRDSSL THKALIHGPA YVGDECFIGF 121 RSTVFNARVG HGCIVMMHAL IQDVEIPPGK YVPSGAIITS 161 QQQADRLPDV RQEDKDFAHH VVGINEALLA GYHCARSSAC 201 INPIRAGLSQ TFQGSTPGTH GLEESINGTT NTMNNGYGLS 241 PALISQVRSL LAQGYRIGTE HATPRRFKTS SWESCAPIES 281 KNEGQVLSAL SGCLQEHQGE YVRLLGIDVQ ARRRVLEVLI 321 QRPDGKPTSL STRGTVSVAA PSASNGHRSS TAGTSNGGGS 361 LADQVRGLLQ QGCRITTEHA DKRRFKTSSW QVGAVIESSN 401 FNQVMAALDS AMQQYSGEYV RLIAVDPLAK RRVAEVLIHR 441 PDGKPVATTA ASKGSTYSSN GASNGASNGA SSNGYGGGSV 481 SGLSGETANQ VRGWLGQGYR ISAEYADKRR FKTGSWQTHG 521 TLEGRGDQVL GSISTVLSTH SGNYVRLVGV DPQAKRRVGQ 561 VIIQRP

A related CcmM protein from Hassallia byssoidea has a sequence has at least 53% sequence identity to SEQ ID NO:1, as illustrated below. Asterisks below the compared sequences indicate amino acid identity at that position (SEQ ID NO:4).

52.5% identity in 541 residues overlap; Score: 1402.0; Gap frequency: 1.1% Seq1 3 SPTTVPVATAGRLAEPYIDPAAQVHAIASIIGDVRIAAGVRVAAGVSIRADEGAPFQVGK Seq4 5 STAAPPTPWSRNLAEPNIDATAYIHPFSNVIGDVRIGANVIVAPGTSIRADEGTPFNISE *    *      **** **  *  *     ****** * * ** * ******* ** Seq1 63 ESILQEGAVIHGLEYGRVLGDDQADYSVWIGQRVAITHKALIHGPAYLGDDCFVGFRSTV Seq4 65 NTNLQDGVVIHGLEQGRVIGDDDNQYSVWIGKNASITHMALIHGPAYVGDDCFIGFRSTV    ** * ****** *** ***   ******    *** ******** ***** ****** Seq1 123 FNARVGAGSVIMMHALVQDVEIPPGRYVPSGAIITTQQQADRLPEVRPEDREFARHIIGS Seq4 125 FNARVGNGCIVMMHALIQDVEIPPGKYVPSGAIITNQQQADRLPDVQVQDREFSHHVVGI ****** *   ***** ******** ********* ******** *   ****  *  * Seq1 183 PPVIVRSTPAATADFHSTPTPSPLRPSSSEATTVSAYNGQG----RLSSEVITQVRSLLN Seq4 185 NQAL-RSGYLCAADNKCIKNIRNEMTSSYKTNGSNGYSGNGSVSSNLSSETVQQVRHLLE      **     **            **        * * *     ****   *** ** Seq1 239 QGYRIGTEHADKRRFRTSSWQPCAPIQSTNERQVLSELENCLSEHEGEYVRLLGIDTNTR Seq4 244 QGYQIGTEHVDQRRFRTGSWASCSPIATNSTSEAIAALESCLAEHSGEFVRLFGIDPKGK *** ***** * ***** **  * **           ** ** ** ** *** *** Seq1 299 SRVFEALIQRPDGSVPESLGSQPVAVASGGGRQSSYASVSGNLSAEVVNKVRNLLAQGYR Seq4 304 RRVLETIIQRPDGVVQNGT-TPKLGVKSASYSGGNSYSGSSTLSGEAIEQVRQLLAGGYK  ** *  ****** *          * *         * *  ** *    ** *** ** Seq1 359 IGTEHADKRRFRTSSWQSCAPIQSSNERQVLAELENCLSEHEGEYVRLLGIDTASRSRVF Seq4 363 IGMEHVDKRRFRTGSWQSCTPIASSNEKEVISALEACVASHTGEYVRLVGIEPKARKRVL ** ** ******* ***** ** ****  *   ** *   * ****** **    * ** Seq1 419 EALIQDPQGPVGSAKAAAAPVSSATPSSHSYTSNGSSSSDVAGQVRGLLAQGYRISAEVA Seq4 423 ESIIQRPDGNVAEGSSNKFVASSSSESRTSTNASTRLSPEVIDQLRQLINQGSKISAEHV *  ** * * *          **   *  *       *  *  * * *  **  **** Seq1 479 DKRRFQTSSWQSLPALSGQSEATVLPALESILQEHKGKYVRLIGIDPAARRRVAELLIQK Seq4 483 DKRRFRTGSWASCGQIQGNSEREAIAALEGYLREYQGEYVRLIGIEPKAKKRVLESIIQR ***** * ** *     * **     ***  * *  * ******* * *  ** *  ** Seq1 539 P Seq4 543 P * This related CcmM protein from Hassallia byssoidea has a sequence that is available from the National Center for Biotechnology Information database (see website at ncbi.nlm.nih.gov) with accession number WP_039748670.1 (GI:748175120), and with the sequence shown below (SEQ ID NO:4).

1 MAVRSTAAPP TPWSRNLAEP NIDATAYIHP FSNVIGDVRI 41 GANVIVAPGT SIRADEGTPF NISENTNLQD GVVIHGLEQG 81 RVIGDDDNQY SVWIGKNASI THMALIHGPA YVGDDCFIGF 121 RSTVFNARVG NGCIVMMHAL IQDVEIPPGK YVPSGAIITN 161 QQQADRLPDV QVQDREFSHH VVGINQALRS GYLCAADNKC 201 IKNIRNEMTS SYKTNGSNGY SGNGSVSSNL SSETVQQVRH 241 LLEQGYQIGT EHVDQRRFRT GSWASCSPIA TNSTSEAIAA 281 LESCLAEHSG EFVRLFGIDP KGKRRVLETI IQRPDGVVQN 321 GTTPKLGVKS ASYSGGNSYS GSSTLSGEAI EQVRQLLAGG 361 YKIGMEHVDK RRFRTGSWQS CTPIASSNEK EVISALEACV 401 ASHTGEYVRL VGIEPKARKR VLESIIQRPD GNVAEGSSNK 441 FVASSSSESR TSTNASTRLS PEVIDQLRQL INQGSKISAE 481 HVDKRRFRTG SWASCGQIQG NSEREAIAAL EGYLREYQGE 521 YVRLIGIEPK AKKRVLESII QRPDDSVAQS SRSDNQVVAS 561 SSSSTSKTSN TATSTRLSSE VVDQLRQLRN QGSKISVEHV 601 DQRRFRTGSW TSGGQIQGNS EREAIAALEG YLREYEGEYV 641 RLIGINPKDK RRVLETIIQR P

CcmM comprises an N-terminal γ-CA domain followed by three small subunit-like domains (SSLDs) with sequence homology to RbcS, the small subunit of Rubisco (Long et al., 2007).

M35 Protein

The ccmM gene encodes two essential carboxysome components, the full-length protein and a truncated form containing only the SSLDs (known as M35 in Synechococcus elongatus PCC 7942). In Synechococcus, the short form is composed of three SSLDs, which are believed to aggregate Rubisco. An amino acid sequence for the CcmM short form from Synechococcus elongatus PCC 7942 is shown below as SEQ ID NO:5, where the SSLD domains are identified in bold and with underlining.

215      TVSAYN GQGRLSSEVI TQVRSLLNQG YRIGTEHADK 251 RRFRTSSWQP CAPIQSTNER QVLSELENCL SEHEGEYVRL 291 LGIDTNTRSR VFEALIQRP D GSVPESLGSQ PVAVASGGGR 331 QSSYASVSGN L SAEVVNKVR NLLAQGYRIG TEHADKRRFR 371 TSSWQSCAPI QSSNERQVLA ELENCLSEHE GEYVRLLGID 411 TASRSRVFEA LIQDP QGPVG SAKAAAAPVS SATPSSHSYT 451 SNGS SSSDVA GQVRGLLAQG YRISAEVADK RRFQTSSWQS 491 LPALSGQSEA TVLPALESIL QEHKGKYVRL IGIDPAARRR 531 VAELLIQKP As illustrated, an SSLD can include any of SEQ ID NOs:75-77.

(SEQ ID NO: 75) 215     TVSAYN GQGRLSSEVI TQVRSLLNQG YRIGTEHADK 251 RRFRTSSWQP CAPIQSTNER QVLSELENCL SEHEGEYVRL 291 LGIDTNTRSR VFEALIQRP (SEQ ID NO: 76) 331             SAEVVNKVR NLLAQGYRIG TEHADKRRFR 371 TSSWQSCAPI QSSNERQVLA ELENCLSEHE GEYVRLLGID 411 TASRSRVFEA LIQDP (SEQ ID NO: 77) 451     SSSDVA GQVRGLLAQG YRISAEVADK RRFQTSSWQS 491 LPALSGQSEA TVLPALESIL QEHKGKYVRL IGIDPAARRR 531 VAELLIQKP

Some forms of M35 in other cyanobacteria (also referred to as a short form CcmM) can have more SSLDs and can be of varying lengths. Some forms of M35 can have a few amino acids missing from the N-terminus or the C-terminus when compared to the Synechococcus elongatus PCC 7942 M35 protein. For example, there can be one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten few amino acids missing from the N-terminus or the C-terminus of some forms of M35 when compared to the Synechococcus elongatus PCC 7942 M35 protein. In addition, the N-terminus of M35 proteins can have a methionine.

A related short form CcmM protein from Acaryochloris marina has a sequence has at least 56% sequence identity to SEQ ID NO:5, as illustrated below (where the SEQ ID NO:5 sequence is the Query protein).

57.1% identity in 326 residues overlap; Score: 877.0; Gap frequency: 4.0% Seq5 11 LSSEVITQVRSLLNQGYRIGTEHADKRRFRTSSWQPCAPIQSTNERQVLSELENCLSEHE Seq6 228 LDAAIVSQVRSLLAQGYRIGSEHADKRRFQTSSWQSCPSITSTNESQVLAGIESCMSEHQ  *      ****** ****** ******** ***** *  * **** ***   * * *** Seq5 71 GEYVRLLGIDTNTRSRVFEALIQRPDGSVPESLGSQPVAVASGGGRQSSYASVSGNLSAE Seq6 288 GEYVRLIGIDTQARQRVLETIIQRPDGPVKSASISSVTKTIK--NYTTSHISSSGNIDAE ****** ****  * ** *  ****** *     *             *  * ***  ** Seq5 131 VVNKVRNLLAQGYRIGTEHADKRRFRTSSWQSCAPIQSSNERQVLAELENCLSEHEGEYV Seq6 346 TIAHVRSLLGQGYRIGTEHADARRFQTSSWQSCSPIASQQESQVVAALEACIVEHQGEYV     ** ** *********** *** ******* ** *  * ** * ** *  ** **** Seq5 191 RLLGIDTASRSRVFEALIQDPQGPVGSAKAAAAPVSSATPSSH----SYTSNGSSS---- Seq6 406 RMLGIDTQAKQRVFEAIIQRPSDKPKAAPKASRPASTSSSSSSYASPSYASSSPNSGTST * *****    ***** ** *      *  *  * *    **     ** *    * Seq5 243 ---SDVAGQVRGLLAQGYRISAEVADKRRFQTSSWQSLPALSGQSEATVLPALESILQEH Seq6 466 GLGADAIAQVRSLLAQGYRVGYEYADKRRFQTSSWQSCTPINSQQESQVIAALESCIAEH     *   *** *******   * *************      * *  *  ****   ** Seq5 300 KGKYVRLIGIDPAARRRVAELLIQKP Seq6 526 PGNYVRLIGIDPKAKRRVLEVIIQRP  * ********* * *** *  ** * This related protein from Acaryochloris marina has a sequence that is available from the National Center for Biotechnology Information database (see website at ncbi.nlm.nih.gov) with accession number WP_012165581.1 (GI:501116295), and with the full length sequence shown below (SEQ ID NO:6).

1 MVIHSPSTSA SMQAGNLPDP RVSSSAYVHS FAKVMGDVHV 41 GANALIAPGS TIQADQGLPF HIGDNVNIQD GAVIHAIEPG 81 QVRGKDGQNY AVWIGNNSCV THMALIHGPA FIGDNCFIGF 121 RSTVFNAKVG DNCVIMMHAL IQGVEIPPGK YVPSGAVITK 161 QEQANLLPDV LESDRKFTQQ IIHVNEALKS EISGASTKTS 201 IRPARANIGH SQSHRFTTDT KPMNHTTLDA AIVSQVRSLL 241 AQGYRIGSEH ADKRRFQTSS WQSCPSITST NESQVLAGIE 281 SCMSEHQGEY VRLIGIDTQA RQRVLETIIQ RPDGPVKSAS 321 ISSVTKTIKN YTTSHISSSG NIDAETIAHV RSLLGQGYRI 361 GTEHADARRF QTSSWQSCSP IASQQESQVV AALEACIVEH 401 QGEYVRMLGI DTQAKQRVFE AIIQRPSDKP KAAPKASRPA 441 STSSSSSSYA SPSYASSSPN SGTSTGLGAD AIAQVRSLLA 481 QGYRVGYEYA DKRRFQTSSW QSCTPINSQQ ESQVIAALES 521 CIAEHPGNYV RLIGIDPKAK RRVLEVIIQR PDSNSKASPS 561 APKARPASSS SSYSSKVESN SSSYRPAPSA GLDGTVVNQI 601 RSLLAQGYRI GTEYADKRRF QTSSWQSCTP IASQQESQVI 641 AGVEACMAEH PNDYVRLIGI DKRAKRRMSE TIIQRPGGST 681 ATSSSVKTSS SRSYQAPAAK SSRGRGFSPR NGGSLDADTV 721 AQVRSLLAQG YRISTEYADK RRFQTSSWQS CPPIKTQQES 761 QVIAALESCM ADHQKEYVRL IGIDTNAKRR VLESVIQKPV 801 AAH The short form CcmM portion of this Acaryochloris marina related protein contains five SSLDs and is shown below as SEQ ID NO:7.

                      KPMNHTTLDA AIVSQVRSLL 241 AQGYRIGSEH ADKRRFQTSS WQSCPSITST NESQVLAGIE 281 SCMSEHQGEY VRLIGIDTQA RQRVLETIIQ RPDGPVKSAS 321 ISSVTKTIKN YTTSHISSSG NIDAETIAHV RSLLGQGYRI 361 GTEHADARRF QTSSWQSCSP IASQQESQVV AALEACIVEH 401 QGEYVRMLGI DTQAKQRVFE AIIQRPSDKP KAAPKASRPA 441 STSSSSSSYA SPSYASSSPN SGTSTGLGAD AIAQVRSLLA 481 QGYRVGYEYA DKRRFQTSSW QSCTPINSQQ ESQVIAALES 521 CIAEHPGNYV RLIGIDPKAK RRVLEVIIQR PDSNSKASPS 561 APKARPASSS SSYSSKVESN SSSYRPAPSA GLDGTVVNQI 601 RSLLAQGYRI GTEYADKRRF QTSSWQSCTP IASQQESQVI 641 AGVEACMAEH PNDYVRLIGI DKRAKRRMSE TIIQRPGGST 681 ATSSSVKTSS SRSYQAPAAK SSRGRGFSPR NGGSLDADTV 721 AQVRSLLAQG YRISTEYADK RRFQTSSWQS CPPIKTQQES 761 QVIAALESCM ADHQKEYVRL IGIDTNAKRR VLESVIQKPV 801 AAH Some forms of CcmM can have a few amino acids missing from the N-terminus or the C-terminus of the short form CcmM protein. In addition, the N-terminus of the short form CcmM protein can have a methionine.

A related short form CcmM protein from Thermosynechococcus elongatus BP-1 has a sequence has at least 49% sequence identity to SEQ ID NO:5, as illustrated below.

48.1% identity in 316 residues overlap; Score: 685.0; Gap frequency: 3.8% Seq5 11 LSSEVITQVRSLLNQGYRIGTEHADKRRFRTSSWQPCAPIQSTNERQVLSELENCLSEHE Seq8 229 MTTDYGTHVRQLLQQGYQISLEYADARRYRTSSWQSGPTLTGQQESQVMAAIAQLLKEHE       * ** ** *** *  * ** ** ******         * **       * *** Seq5 71 GEYVRLLGIDTNTRSRVFEALIQRP-DGSVPESLGSQPVAVASGGGRQSSYASVSGNLSA Seq8 289 GEYVRLIGVDPKAKRRVFEEIIQRPGQAAVASSSSSRPSATVN--------ASPVGSLDA ****** * *     ****  ****    *  *  * * *           **  * * * Seq5 130 EVVNKVRNLLAQGYRIGTEHADKRRFRTSSWQSCAPIQSSNERQVLAELENCLSEHEGEY Seq8 341 AVVAQVRQLLQQGYQIGTEHADARRYRTSSWTSCAPIQSKQEPEVLAALEACLQEHAGEY  **  ** ** *** ******* ** ***** *******  *  *** ** ** ** *** Seq5 190 VRLLGIDTASRSRVFEALIQDPQGPVGSAKAAAAPVSSATPSSHSYTSNGSSSSDVAGQV Seq8 401 VRLIGIDQKQKRRVLEQIIQRPQGPVAIAPKTPTPVATSHASVSSGGNDTLLSADLVNQI *** ***     ** *  ** *****  *     **     *  *       * *   * Seq5 250 RGLLAQGYRISAEVADKRRFQTSSWQSLPALSGQSEATVLPALESILQEHKGKYVRLIGI Seq8 461 QDLLRQGCQVITEYADQRRFRTSSWQSGIKITSAQQ---INDLRSFLAEHQRDYIRLVGV   ** **     * ** *** ******               * * * **   * ** * Seq5 310 DPAARRRVAELLIQKP Seq8 518 NPQAKQRVLETIIHRP  * *  ** *  *  * This related protein from Thermosynechococcus elongatus BP-1 has a sequence that is available from the National Center for Biotechnology Information database (see website at ncbi.nlm.nih.gov) with accession number NP_681734.1 (GI:22298487) and with the sequence shown below (SEQ ID NO:8).

1 MAVQSYAAPP TPWSRDLAEP EIAPTAYVHS FSNLIGDVRI 41 KDYVHIAPGT SIRADEGTPF HIGSRTNIQD GVVIHGLQQG 81 RVIGDDGQEY SVWIGDNVSI THMALIHGPA YIGDGCFIGF 121 RSTVFNARVG AGCVVMMHVL IQDVEIPPGK YVPSGMVITT 161 QQQADRLPNV EESDIHFAQH VVGINEALLS GYQCAENIAC 201 IAPIRNELQR QEDPPTLHVE MLTGEKNTMT TDYGTHVRQL 241 LQQGYQISLE YADARRYRTS SWQSGPTLTG QQESQVMAAI 281 AQLLKEHEGE YVRLIGVDPK AKRRVFEEII QRPGQAAVAS 321 SSSSRPSATV NASPVGSLDA AVVAQVRQLL QQGYQIGTEH 361 ADARRYRTSS WTSCAPIQSK QEPEVLAALE ACLQEHAGEY 401 VRLIGIDQKQ KRRVLEQIIQ RPQGPVAIAP KTPTPVATSH 441 ASVSSGGNDT LLSADLVNQI QDLLRQGCQV ITEYADQRRF 481 RTSSWQSGIK ITSAQQINDL RSFLAEHQRD YIRLVGVNPQ 521 AKQRVLETII HRPNGKAASN GNSTRGQGFT PRPTASSQGS 561 PSTHSLSQEV IEQVRQLLQQ GYTLGLEHVD ARRYRTNSWQ 601 SGPRIEAKNL NEALAAIQAC LQEYSGEYVR LIGINPAGKQ 641 RVAEILLQQA AK The short form CcmM portion of this Thermosynechococcus elongatus BP-1 related protein has four SSLDS and is shown below as SEQ ID NO:9.

201                                      THVRQL 241 LQQGYQISLE YADARRYRTS SWQSGPTLTG QQESQVMAAI 281 AQLLKEHEGE YVRLIGVDPK AKRRVFEEII QRPGQAAVAS 321 SSSSRPSATV NASPVGSLDA AVVAQVRQLL QQGYQIGTEH 361 ADARRYRTSS WTSCAPIQSK QEPEVLAALE ACLQEHAGEY 401 VRLIGIDQKQ KRRVLEQIIQ RPQGPVAIAP KTPTPVATSH 441 ASVSSGGNDT LLSADLVNQI QDLLRQGCQV ITEYADQRRF 481 RTSSWQSGIK ITSAQQINDL RSFLAEHQRD YIRLVGVNPQ 521 AKQRVLETII HRPNGKAASN GNSTRGQGFT PRPTASSQGS 561 PSTHSLSQEV IEQVRQLLQQ GYTLGLEHVD ARRYRTNSWQ 601 SGPRIEAKNL NEALAAIQAC LQEYSGEYVR LIGINPAGKQ 641 RVAEILLQQA AK Some short forms of CcmM can have a few amino acids missing from the N-terminus or the C-terminus of the M35 protein. In addition, the N-terminus of the short form protein can have a methionine.

A related short form CcmM protein from Trichormus azollae has a sequence has at least 52% sequence identity to SEQ ID NO:5, as illustrated below.

51.1% identity in 321 residues overlap; Score: 798.0; Gap frequency: 1.9% Seq5 10 RLSSEVITQVRSLLNQGYRIGTEHADKRRFRTSSWQPCAPIQSTNERQVLSELENCLSEH Seq10 233 KLGAEIVDQVRYLLNQGYKIGTEHVDQRRFRTGSWQSCQPIETRSLGEAITALESCLIDH  *  *   *** ****** ***** * ***** *** * **           ** **  * Seq5 70 EGEYVRLLGIDTNTRSRVFEALIQRPDGSVPESLGSQPVAVAS----GGGRQSSYASVSG Seq10 293 SGEYVRLFGID-NGRKRVLETIIQRPDGVVATSTSSFKTPAASYSSYNGNGNSNGAVASG  ****** *** * * ** *  ****** *  *  *     **     *   *  *  ** Seq5 126 NLSAEVVNKVRNLLAQGYRIGTEHADKRRFRTSSWQSCAPIQSSNERQVLAELENCLSEH Seq10 352 SLSAETVNQIRQLLANGYKIGTEHVDQRRFRTGSWQSCNPIEATSANDVVAALEECMTSH  **** **  * *** ** ***** * ***** ***** **       * * ** *   * Seq5 186 EGEYVRLLGIDTASRSRVFEALIQDPQGPVGSAKAAAAPVSSATPSSHSYTSNGSS-SSD Seq10 412 QGEYVRLIGIDSKAKRRVLEAIIQRPNGQVVSSGSAKTSGTLYSGATASATATSTRLSTE  ****** ***     ** ** ** * * * *   *            * *      * Seq5 245 VAGQVRGLLAQGYRISAEVADKRRFQTSSWQSLPALSGQSEATVLPALESILQEHKGKYV Seq10 472 VVDQLKQLLTGGFKISVEHVDQRRFRTGSWVSCGQIQATSERDVLAALEAVISEYAGEYV *  *   **  *  ** *  * *** * ** *      **  ** ***    *  * ** Seq5 305 RLIGIDPAARRRVAELLIQKP Seq10 532 RLIGIDPVAKRRVLEAIIQRP ******* * *** *  ** * This short form CcmM related protein from Trichormus azollae has a sequence that is available from the National Center for Biotechnology Information database (see website at ncbi.nlm.nih.gov) with accession number WP_013190978.1 (GI:502956002), and with the sequence shown below (SEQ ID NO:10).

1 MVVRSTAAPP TPWSRSLAEP DIHQTAFVHS SCNLIGDVHL 41 GQNVIIAPGT SIRADEGTPF FIGENTNIQD GVVIHGLEQG 81 RVIGDDGKNY SVWVGKDASI THMALIHGPA YVGESCFIGF 121 RSTVFNARVG AGCIVMMHAL IQDVEIPPGK YVASGSIITM 161 QQQADRLPDV QAQDQQFAHH VVGINQALRA GYRCVEDIKC 201 IAPIRDELNL SGDRSYTSII VDELERSSEV ASKLGAEIVD 241 QVRYLLNQGY KIGTEHVDQR RFRTGSWQSC QPIETRSLGE 281 AITALESCLI DHSGEYVRLF GIDNGRKRVL ETIIQRPDGV 321 VATSTSSFKT PAASYSSYNG NGNSNGAVAS GSLSAETVNQ 361 IRQLLANGYK IGTEHVDQRR FRTGSWQSCN PIEATSANDV 401 VAALEECMTS HQGEYVRLIG IDSKAKRRVL EAIIQRPNGQ 441 VVSSGSAKTS GTLYSGATAS ATATSTRLST EVVDQLKQLL 481 TGGFKISVEH VDQRRFRTGS WVSCGQIQAT SERDVLAALE 521 AVISEYAGEY VRLIGIDPVA KRRVLEAIIQ RP The short form portion of this Trichormus azollae related protein contains three SSLDs and is shown below as SEQ ID NO:11.

233                                V ASKLGAEIVD 241 QVRYLLNQGY KIGTEHVDQR RFRTGSWQSC QPIETRSLGE 281 AITALESCLI DHSGEYVRLF GIDNGRKRVL ETIIQRPDGV 321 VATSTSSFKT PAASYSSYNG NGNSNGAVAS GSLSAETVNQ 361 IRQLLANGYK IGTEHVDQRR FRTGSWQSCN PIEATSANDV 401 VAALEECMTS HQGEYVRLIG IDSKAKRRVL EAIIQRPNGQ 441 VVSSGSAKTS GTLYSGATAS ATATSTRLST EVVDQLKQLL 481 TGGFKISVEH VDQRRFRTGS WVSCGQIQAT SERDVLAALE 521 AVISEYAGEY VRLIGIDPVA KRRVLEAIIQ RP Some short forms of CcmM can have a few amino acids missing from the N-terminus or the C-terminus of the protein. In addition, the N-terminus of the short form CcmM protein can have a methionine.

CcmN Protein—Encapsulation Peptide (EP)

CcmN contains multiple hexapeptide-repeats and, at its C-terminus, an encapsulation peptide (EP), which is a short α-helical segment linked to the hexapeptide-repeat domains by a flexible linker sequence (Kinney et al., 2012). In general, encapsulation peptides have poorly conserved sequences but are amphipathic in nature (Aussignargues et al., 2015) A schematic diagram of the CcmN protein is shown in FIG. 1A.

An amino acid sequence for a Synechococcus elongatus PCC 7942 carbon dioxide concentrating mechanism protein (CcmN: Synpcc7942_1424) is available as accession number ABB57454 (SEQ ID NO:12).

1 MHLPPLEPPI SDRYFASGEV TIAADVVIAP GVLLIAEADS 41 RIEIASGVCI GLGSVIHARG GAIIIQAGAL LAAGVLIVGQ 81 SIVGRQACLG ASTTLVNTSI EAGGVTAPGS LLSAETPPTT 121 ATVSSSEPAG RSPQSSAIAH PTKVYGKEQF LRMRQSMFPD 161 R

As illustrated herein, SSLDs domains are fused with an encapsulation peptide from a CcmN protein. Such an encapsulation peptide can have the following sequence (SEQ ID NO:13).

1 VYGKEQFLRM RQSMFPDR

A related CcmN encapsulation peptide is available from Prochlorothrix hollandica that has at least 65% sequence identity to SEQ ID NO:13, as illustrated below.

Score Expect Identities Positives Gaps 30.8 bits 0.32 11/17 (65%) 13/17 (76%) 0/17 (0%) (65) Seq 13 VYGKEQFLRMRQSMFPD 17 Seq 14 VYGRDYFLQMRFSLFPD 414 ***   ** ** * *** This Prochlorothrix hollandica related encapsulation peptide has the following sequence: VYGRDYFLQMRFSLFPD (SEQ ID NO:14).

A related CcmN encapsulation peptide is available from Halothece sp. PCC 7418 (Cai et al, 2016) that has at least 27% sequence identity to SEQ ID NO:13, as illustrated below.

Seq13 VYGKEQFLRMRQSMFPDR-------------------------- 18 Seq15 IYGQTHIERLMVTLFPHKEKFKKKTNDWFLVLGSLLFDDFPNNE 44 :**: :: *:  ::**.: The Halothece sp. PCC 7418 related encapsulation peptide has the following sequence: IYGQTHIERLMVTLFPHKEKFKKKTNDWFLVLGSLLFDDFPNNE (SEQ ID NO:15).

A related CcmN encapsulation peptide is available from Moorea producens that has at least 56% sequence identity to SEQ ID NO:13, as illustrated below.

Score Expect Identities Positives Gaps 28.2 bits 2.6 10/18 (56%) 12/18 (66%) 4/18 (22%) (59) Seq13 EQFLR-MRQSM---FPDR 18 Seq16 606 EQFFRRMRQSLNRAFSER 623 *** * ****    *  * The Moorea producens related encapsulation peptide has the following sequence: EQFFRRMRQSLNRAFSER (SEQ ID NO:16).

CcaA Carbonate Dehydratase (Carbonic Anhydrase)

While the CcmM and CcmN are typically conserved and are needed for native carboxysome formation (Long et al., 2010; Kinney et al., 2012), CcaA deletion mutant cell lines can still form carboxysomes (So et al., 2002b). Such CcaA deletion mutant cells exhibit a high carbon dioxide-requiring (hcr) phenotype. The CcaA genes encode carbonic anhydrase, also called carbonate dehydratase. A schematic diagram of the carbonate dehydratase, CcaA, protein is shown in FIG. 1A.

An amino acid sequence for a Synechococcus elongatus PCC 7942 carbonate dehydratase (CcaA; Synpcc7942_1447; 30185 daltons) is available as accession number ABB57477.1 (see website at uniprot.org/uniprot/P27134)(SEQ ID NO:17).

1 MRKLIEGLRH FRTSYYPSHR DLFEQFAKGQ HPRVLFITCS 41 DSRIDPNLIT QSGMGELFVI RNAGNLIPPF GAANGGEGAS 81 IEYAIAALNI EHVVVCGHSH CGAMKGLLKL NQLQEDMPLV 121 YDWLQHAQAT RRLVLDNYSG YETDDLVEIL VAENVLTQIE 161 NLKTYPIVRS RLFQGKLQIF GWIYEVESGE VLQISRTSSD 201 DTGIDECPVR LPGSQEKAIL GRCVVPLTEE VAVAPPEPEP 241 VIAAVAAPPA NYSSRGWLAP EQQQRIYRGN AS

A related CcaA carbonate dehydratase is available from Synechococcus elongatus that has at least 99% sequence identity to SEQ ID NO:17, as illustrated below.

99.6% identity in 272 residues overlap; Score: 1415.0; Gap frequency: 0.0% Seq17 1 MRKLIEGLRHFRTSYYPSHRDLFEQFAKGQHPRVLFITCSDSRIDPNLITQSGMGELFVI Seq18 1 MRKLIEGLRHFRTSYYPSHRDLFEQFAKGQHPRVLFITCSDSRIDPNLITQSGMGELFVI ************************************************************ Seq17 61 RNAGNLIPPFGAANGGEGASIEYAIAALNIEHVVVCGHSHCGAMKGLLKLNQLQEDMPLV Seq18 61 RNAGNLIPPFGAANGGEGASIEYAIAALNIEHVVVCGHSHCGAMKGLLKLNQLQEDMPLV ************************************************************ Seq17 121 YDWLQHAQATRRLVLDNYSGYETDDLVEILVAENVLTQIENLKTYPIVRSRLFQGKLQIF Seq18 121 YDWLQHAQATRRLVLDNYSGYETDDLVEFLVAENVLTQIENLKTYPIVRSRLFQGKLQIF **************************** ******************************* Seq17 181 GWIYEVESGEVLQISRTSSDDTGIDECPVRLPGSQEKAILGRCVVPLTEEVAVAPPEPEP Seq18 181 GWIYEVESGEVLQISRTSSDDTGIDECPVRLPGSQEKAILGRCVVPLTEEVAVAPPEPEP ************************************************************ Seq17 241 VIAAVAAPPANYSSRGWLAPEQQQRIYRGNAS Seq18 241 VIAAVAAPPANYSSRGWLAPEQQQRIYRGNAS ******************************** This CcaA related protein from Synechococcus elongatus has a sequence that is available from the National Center for Biotechnology Information database (see website at ncbi.nlm.nih.gov) with accession number WP_011242423.1 (GI:499561640), and with the sequence shown below (SEQ ID NO:18).

1 MRKLIEGLRH FRTSYYPSHR DLFEQFAKGQ HPRVLFITCS 41 DSRIDPNLIT QSGMGELFVI RNAGNLIPPF GAANGGEGAS 81 IEYAIAALNI EHVVVCGHSH CGAMKGLLKL NQLQEDMPLV 121 YDWLQHAQAT RRLVLDNYSG YETDDLVEFL VAENVLTQIE 161 NLKTYPIVRS RLFQGKLQIF GWIYEVESGE VLQISRTSSD 201 DTGIDECPVR LPGSQEKAIL GRCVVPLTEE VAVAPPEPEP 241 VIAAVAAPPA NYSSRGWLAP EQQQRIYRGN AS

A related CcaA carbonate dehydratase is available from Geminocystis herdmanii that has at least 55% sequence identity to SEQ ID NO:17, as illustrated below.

58.3% identity in 278 residues overlap; Score: 779.0; Gap frequency: 2.9% Seq17 1 MRKLIEGLRHFRTSYYPSHRDLFEQFAKGQHPRVLFITCSDSRIDPNLITQSGMGELFVI Seq19 1 MKKIIEGLHRFQAGYFESHRDLFEQLSHGQHPRILFITCSDSRIDPNLITQANVGELFVI * * ****  *   *  ********   ***** *****************   ****** Seq17 61 RNAGNLIPPFGAANGGEGASIEYAIAALNIEHVVVCGHSHCGAMKGLLKLNQLQEDMPLV Seq19 61 RNAGNIIPPFGATNGGEGASIEYAITALDIEQVIVCGHSHCGAMKGLLKMSKLADKMPLV ***** ****** ************ ** ** * ***************   *   **** Seq17 121 YDWLQHAQATRRLVLDNYSGYETDDLVEILVAENVLTQIENLKTYPIVRSRLFQGKLQIF Seq19 121 YEWLKQAEATRRLIIDNYSHLEGEELLQITVAENVLTQLENLNTYPIVRSRLHQGRLSLH * **  * *****  ****  *   *  * ******** *** ********* ** * Seq17 181 GWIYEVESGEVLQISRTSSDDTGID------ECPVRLPGSQEKAILGRCVVPLTEEVAVA Seq19 181 GWIYGIETGEVLTYDPKVHDFVNLESRTDNSEYIYNLHPSCSVAKSMFYGIPDENDDKVQ ****  * ****       *           *    *  *   *       *      * Seq17 235 PPEPEPVIAAVAAPPANYSSR--GWLAPEQQQRIYRGN Seq19 241 PSEPIPQTINPNLPRSRSGAARSNRLSPEQEQRIYRGS * ** *       *           * *** ****** This CcaA related protein from Geminocystis herdmanii has a sequence that is available from the National Center for Biotechnology Information database (see website at ncbi.nlm.nih.gov) with accession number WP_017295030.1 (GI:515864402), and with the sequence shown below (SEQ ID NO:19).

1 MKKIIEGLHR FQAGYFESHR DLFEQLSHGQ HPRILFITCS 41 DSRIDPNLIT QANVGELFVI RNAGNIIPPF GATNGGEGAS 81 IEYAITALDI EQVIVCGHSH CGAMKGLLKM SKLADKMPLV 121 YEWLKQAEAT RRLIIDNYSH LEGEELLQIT VAENVLTQLE 161 NLNTYPIVRS RLHQGRLSLH GWIYGIETGE VLTYDPKVHD 201 FVNLESRTDN SEYIYNLHPS CSVAKSMFYG IPDENDDKVQ 241 PSEPIPQTIN PNLPRSRSGA ARSNRLSPEQ EQRIYRGST

A related CcaA carbonate dehydratase is available from Aliterella atlantica that has at least 74% sequence identity to SEQ ID NO:17, as illustrated below.

57.2% identity in 271 residues overlap; Score: 786.0; Gap frequency: 0.4% Seq17 1 MRKLIEGLRHFRTSYYPSHRDLFEQFAKGQHPRVLFITCSDSRIDPNLITQSGMGELFVI Seq20 1 MRKLIKGLRAFKDNYYSNHLELFEKLTHAQKPRVLFITCSDSRIDPNLITQAAVGELFVI ***** *** *   **  *  ***     * ********************   ****** Seq17 61 RNAGNLIPPFGAANGGEGASIEYAIAALNIEHVVVCGHSHCGAMKGLLKLNQLQEDMPLV Seq20 61 RNAGNLIPPFGATNGGEGATVEYAVHALGIEQIVVCGHSHCGAMKGLLKLNKLQQDMPLV ***** ****** ******  ***  ** **  ****************** ** ***** Seq17 121 YDWLQHAQATRRLVLDNYSGYETDDLVEILVAENVLTQIENLKTYPIVRSRLFQGKLQIF Seq20 121 YNWLQYAESTRRLVQENYNSYSEEELVEIAVAENVLTQIENLKTYPVVRSKLYQGKLQIY * *** *  *****  **  *    **** **************** *** * ****** Seq17 181 GWIYEVESGEVLQISRTSSDDTGIDECPVRLPGSQE-KAILGRCVVPLTEEVAVAPPEPE Seq20 181 AWIYHLETGEVLAYDPQSHAYVAPQSQLMNGDTTESIETRIANTSAPIVACEFPSRHKQR  ***  * ****     *                            * Seq17 240 PVIAAVAAPPANYSSRGWLAPEQQQRIYRGN Seq20 241 QVAHNTANNDGDTLPDMWLSPQQAERIYRGS  *    *          ** * *  ***** This CcaA related protein from Aliterella atlantica has a sequence that is available from the National Center for Biotechnology Information database (see website at ncbi.nlm.nih.gov) with accession number WP_045053064.1 (GI:769918643), and with the sequence shown below (SEQ ID NO:20).

1 MRKLIKGLRA FKDNYYSNHL ELFEKLTHAQ KPRVLFITCS 41 DSRIDPNLIT QAAVGELFVI RNAGNIIPPF GATNGGEGAT 81 VEYAVHALGI EQIVVCGHSH CGAMKGLLKL NKLQQDMPLV 121 YNWLQYAEST RRLVQENYNS YSEEELVEIA VAENVLTQIE 161 NLKTYPVVRS KLYQGKLQIY AWIYHLETGE VLAYDPQSHA 201 YVAPQSQLMN GDTTESIETR IANTSAPIVA CEFPSRHKQR 241 QVAHNTANND GDTLPDMWLS PQQAERIYRG SNGNR

A related CcaA carbonate dehydratase is available from Leptolyngbya boryana that has at least 74% sequence identity to SEQ ID NO:17, as illustrated below.

74.5% identity in 192 residues overlap; Score: 794.0; Gap frequency: 0.0% Seq17 1 MRKLIEGLRHFRTSYYPSHRDLFEQFAKGQHPRVLFITCSDSRIDPNLITQSGMGELFVI Seq21 1 MKKLIQGHQQFWESYVPSHLDQLEELSHGQKPRVLFITCSDSRIDPNLITQAGIGELFVI * *** *   *  ** *** *  *    ** ******************** * ****** Seq17 61 RNAGNLIPPFGAANGGEGASIEYAIAALNIEHVVVCGHSHCGAMKGLLKLNQLQEDMPLV Seq21 61 RNAGNIIPPFGAANGGEGAAVEYAIAALDIQQIIVCGHSHCGAMKGLLKLNKLQEDMPLV ***** *************  ******* *    ***************** ******** Seq17 121 YDWLQHAQATRRLVLDNYSGYETDDLVEILVAENVLTQIENLKTYPIVRSRLFQGKLQIF Seq21 121 YDWLKHAEATRRLVKENYSQYSGEELLEITIAENVLTQIENLKTYPVVHSRLYQGKLEIY **** ** ******  *** *    * **  *************** * *** **** * Seq17 181 GWIYEVESGEVL Seq21 181 GWVYHIETGELL ** *  * ** * This CcaA related protein from Leptolyngbya boryana has a sequence that is available from the National Center for Biotechnology Information database (see website at ncbi.nlm.nih.gov) with accession number WP_017285834.1 (GI:515855206), and with the sequence shown below (SEQ ID NO:21).

1 MKKLIQGHQQ FWESYVPSHL DQLEELSHGQ KPRVLFITCS 41 DSRIDPNLIT QAGIGELFVI RNAGNIIPPF GAANGGEGAA 81 VEYAIAALDI QQIIVCGHSH CGAMKGLLKL NKLQEDMPLV 121 YDWLKHAEAT RRLVKENYSQ YSGEELLEIT IAENVLTQIE 161 NLKTYPVVHS RLYQGKLEIY GWVYHIETGE LLAFDPETHA 201 YVPPQSQLSP RELGAFYEKT SAPPVACNLP HKEDNGNGQL 241 RQPVTIRSQV KSAEPVPQTE VMPWLTAEQA QRIYQGSKR

Many cyanobacteria lack CcaA (Zarzycki et al., 2013) and its function can be replaced by the γ-CA domain of CcmM (Peña et al., 2010).

CcmC Chimeric Protein

A streamlined carboxysome core, referred to as CcmC, is described herein that combines segments of several carboxysome components into a single chimeric protein. CcmC contains scaffolding domains (the SSLDs that are involved in nucleating Rubisco), an enzymatic domain (carbonic anhydrase), and an encapsulating domain (the EP). FIG. 1B shows a schematic diagram of the chimeric protein. The following is an amino acid sequence for a CcmC gene (SEQ ID NO:22).

1 MTVSAYNGQG RLSSEVITQV RSLLNQGYRI GTEHADKRRF 41 RTSSWQPCAP IQSTNERQVL SELENCLSEH EGEYVRLLGI 81 DTNTRSRVFE ALIQRPDGSV PESLGSQPVA VASGGGRQSS 121 YASVSGNLSA EVVNKVRNLL AQGYRIGTEH ADKRRFRTSS 161 WQSCAPIQSS NERQVLAELE NCLSEHEGEY VRLLGIDTAS 201 RSRVFEALIQ DPQGPVGSAK AAAAPVSSAT PSSHSYTSNG 241 SSSSDVAGQV RGLLAQGYRI SAEVADKRRF QTSSWQSLPA 281 LSGQSEATVL PALESILQEH KGKYVRLIGI DPAARRRVAE 321 LLIQKPGSRK LIEGLRHFRT SYYPSHRDLF EQFAKGQHPR 361 VLFITCSDSR IDPNLITQSG MGELFVIRNA GNLIPPFGAA 401 NGGEGASIEY AIAALNIEHV VVCGHSHCGA MKGLLKLNQL 441 QEDMPLVYDW LQHAQATRRL VLDNYSGYET DDLVEILVAE 481 NVLTQIENLK TYPIVRSRLF QGKLQIFGWI YEVESGEVLQ 521 ISRTSSDDTG IDECPVRLPG SQEKAILGRC VVPLTEEVAV 561 APPEPEPVIA AVAAPPANYS SRGWLGSGGS VYGKEQFLRM 601 RQSMFPDR Note that amino acids 2-326 of the CcmC protein (with SEQ ID NO:22) are the same as the CcmM short form from Synechococcus elongatus PCC 7942 provided as SEQ ID NO:5. Similarly, amino acids 1-328 of the CcmC protein (with SEQ ID NO:22) are the same as amino acids 1-328 of the M35-EP protein with SEQ ID NO:37. The central amino acids 329-585 of the SEQ ID NO:38 CcmC protein correspond to amino acids 2-258 of the carbonate dehydratase (CcaA) with SEQ ID NO:71. Amino acids 591-608 of the SEQ ID NO:38 CcmC protein correspond to the encapsulation peptide (EP) from a CcmN protein, which has SEQ ID NO:13. Other M35, CcaA, and EP polypeptide segments can substitute for these M35, CcaA, and EP segments to form related CmcC proteins.

Such synthetic CcmC core proteins can support the assembly of functionally competent carboxysomes in cyanobacteria.

Such synthetic CcmC core proteins can have some sequence variation. For example, a CcmC core protein can have at least 40% sequence identity, or at least 50% sequence identity, or at least 60% sequence identity, or at least 70% sequence identity, or at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity sequence identity (or complementarity) with SEQ ID NO:22. Related CcmC proteins can have, for example, 60-99% sequence identity, or 70-99% sequence identity, or 80-99% sequence identity, or 90-95% sequence identity, or 90-99% sequence identity, or 95-97% sequence identity, or 95-98% sequence identity, or 97-99% sequence identity, or 95-99% sequence identity, or 95-100% sequence identity, or 96-100% sequence identity, or 97-100% sequence identity, or 100% sequence identity (or complementarity) with SEQ ID NO:22.

Expression of multiple genes has previously been deemed to be necessary to assemble a BMC core in heterologous systems. However, the construct described herein has a streamlined design that functions to fix carbon even though it is smaller, and consists of a single polypeptide that has small subunit-like domains (SSLDs), Encapsulation peptide (EP), and carbonic anhydrase domains.

The more compact CcmC core protein can accommodate domain components with a variety sequences related to those described herein. For example, a CcmC core protein can have SSLDs (small subunit-like domains), encapsulation peptide (EP), and carbonic anhydrase domains that have at least 40% sequence identity, or at least 50% sequence identity, or at least 60% sequence identity, or at least 70% sequence identity, or at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity, or 60-99% sequence identity, or 70-99% sequence identity, or 80-99% sequence identity, or 90-95% sequence identity, or 90-99% sequence identity, or 95-97% sequence identity, or 97-99% sequence identity, or 100% sequence identity (or complementarity) with any of the SEQ ID NOs described herein.

Previous attempts to engineer bacterial microcompartments have focused on associating heterologous proteins to shell proteins using encapsulation peptides (EPs). For example, through the addition of two different EPs to pyruvate decarboxylase and alcohol dehydrogenase, Lawrence et al. were able to repurpose a propanediol utilization (PDU) compartment for ethanol production (Lawrence et al., 2014). Lin et al. showed that the encapsulation peptide from CcmN targets yellow fluorescent protein into carboxysome-like structures formed in mutant tobacco (Nicotiana benthamiana) plants (Lin et al., 2014b).

In contrast to such previous studies the approach reported here focuses on assembling a multifunctional bacterial microcompartment core using a single polypeptide to nucleate assembly and provide key functions: CcmC nucleates Rubisco, supplies carbonic anhydrase activity, and recruits the shell. This approach allows the packaging of multiple protein domains within a shell using only a single encapsulation peptide (EP).

Shell Proteins

In some cases, it may be useful to express carboxysome shell protein(s) along with the CcmC chimeric core protein.

For example, a carbon dioxide concentrating mechanism protein CcmK and/or CcmL shell protein from Synechococcus elongatus PCC 7942 can be expressed along with the CcmC chimeric core protein. An example of a sequence for such a CcmK shell protein from Synechococcus elongatus PCC 7942 is provided below as SEQ ID NO:23 (see NCBI accession number (ABB56317.1; GI:81167977).

1 MSQQAIGSLE TKGFPPILAA ADAMVKAGRI TIVSYMRAGS 41 ARFAVNIRGD VSEVKTAMDA GIEAAKNTPG GTLETWVIIP 81 RPHENVEAVF PIGFGPEVEQ YRLSAEGTGS GRR

An example of a sequence for such a CcmL shell protein from Synechococcus elongatus PCC 7942 is provided below as SEQ ID NO:24 (see NCBI accession number (ABB57452.1; GI:81169112).

1 MRIAKVRGTV VSTYKEPSLQ GVKFLVVQFL DEAGQALQEY 41 EVAADMVGAG VDEWVLISRG SQARHVRDCQ ERPVDAAVIA 81 IIDTVNVENR SVYDKREHS

Such shell proteins can have some sequence variation. For example, such shell proteins can have at least 40% sequence identity, or at least 50% sequence identity, or at least 60% sequence identity, or at least 70% sequence identity, or at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity, or 60-99% sequence identity, or 70-99% sequence identity, or 80-99% sequence identity, or 90-95% sequence identity, or 90-99% sequence identity, or 95-97% sequence identity, or 97-99% sequence identity, or 100% sequence identity (or complementarity) with SEQ ID NO:23 and/or SEQ ID NO:24.

Rubisco

In some cases, ribulose-1,5-bisphosphate carboxylase/oxygenase, abbreviated as Rubisco herein (also abbreviated as RuBPCase), can also be expressed with the chimeric core carboxysome CcmC protein. Rubisco is an enzyme that can be involved carbon fixation, to provide building blocks for energy-rich molecules such as glucose. Rubisco can catalyze the carboxylation of ribulose-1,5-bisphosphate, and may be one of the most abundant enzymes on Earth.

For example, a ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) protein can be expressed along with the CcmC chimeric core protein. An example of a sequence for such a Rubisco protein from Synechococcus elongatus PCC 7942 is provided below as SEQ ID NO:25 (see NCBI accession number (ABB57456.1; GI:81169116).

1 MPKTQSAAGY KAGVKDYKLT YYTPDYTPKD TDLLAAFRFS 41 PQPGVPADEA GAAIAAESST GTWTTVWTDL LTDMDRYKGK 81 CYHIEPVQGE ENSYFAFIAY PLDLFEEGSV TNILTSIVGN 121 VFGFKAIRSL RLEDIRFPVA LVKTFQGPPH GIQVERDLLN 161 KYGRPMLGCT IKPKLGLSAK NYGRAVYECL RGGLDFTKDD 201 ENINSQPFQR WRDRFLFVAD AIHKSQAETG EIKGHYLNVT 241 APTCEEMMKR AEFAKELGMP IIMHDFLTAG FTANTTLAKW 281 CRDNGVLLHI HRAMHAVIDR QRNHGIHFRV LAKCLRLSGG 321 DHLHSGTVVG KLEGDKASTL GFVDLMREDH IEADRSRGVF 361 FTQDWASMPG VLPVASGGIH VWHMPALVEI FGDDSVLQFG 401 GGTLGHPWGN APGATANRVA LEACVQARNE GRDLYREGGD 441 ILREAGKWSP ELAAALDLWK EIKFEFETMD KL Expression

The chimeric carboxysome core protein, shell protein(s), Rubisco protein(s), and combinations thereof can be expressed from an expression cassette or expression vector. An expression cassette can include a nucleic acid segment that encodes a chimeric carboxysome core protein, shell protein, or Rubisco protein operably linked to a promoter to drive expression. In some cases, such polypeptide(s) can be expressed using convenient vectors, or expression systems. The invention therefore provides expression cassettes or vectors useful for expressing one or more chimeric carboxysome core protein, shell protein, Rubisco protein.

For example, a nucleotide sequence that encodes the chimeric core carboxysome CcmC protein and that can be expressed in a variety of organisms, including Synechococcus elongatus PCC 7942, is shown below as SEQ ID NO:26.

1 ATGACCGTGA GCGCTTATAA CGGCCAAGGC CGACTCAGTT 41 CCGAAGTCAT CACCCAAGTC CGGAGTTTGC TGAACCAGGG 81 CTATCGGATT GGGACGGAAC ATGCGGACAA GCGCCGCTTC 121 CGGACTAGCT CTTGGCAGCC CTGCGCGCCG ATTCAAAGCA 161 CGAACGAGCG CCAGGTCTTG AGCGAACTGG AAAATTGTCT 201 GAGCGAACAC GAAGGTGAAT ACGTTCGCTT GCTCGGCATC 241 GATACCAATA CTCGCAGCCG TGTTTTTGAA GCCCTGATTC 281 AACGGCCCGA TGGTTCGGTT CCTGAATCGC TGGGGAGCCA 321 ACCGGTGGCA GTCGCTTCCG GTGGTGGCCG TCAGAGCAGC 361 TATGCCAGCG TCAGCGGCAA CCTCTCAGCA GAAGTGGTCA 401 ATAAAGTCCG CAACCTCTTA GCCCAAGGCT ATCGGATTGG 441 GACGGAACAT GCAGACAAGC GCCGCTTTCG GACTAGCTCT 481 TGGCAGTCCT GCGCACCGAT TCAAAGTTCG AATGAGCGCC 521 AGGTTCTGGC TGAACTGGAA AACTGTCTGA GCGAGCACGA 561 AGGTGAGTAC GTTCGCCTGC TGGGCATCGA CACTGCTAGC 601 CGCAGTCGTG TTTTTGAAGC CCTGATCCAA GATCCCCAAG 641 GACCGGTGGG TTCCGCCAAA GCGGCCGCCG CACCTGTGAG 681 TTCGGCAACG CCCAGCAGCC ACAGCTACAC CTCAAATGGA 721 TCGAGTTCGA GCGATGTCGC TGGACAGGTT CGGGGTCTGC 761 TAGCCCAAGG CTACCGGATC AGTGCGGAAG TCGCCGATAA 801 GCGTCGCTTC CAAACCAGCT CTTGGCAGAG TTTGCCGGCT 841 CTGAGTGGCC AGAGCGAAGC AACTGTCTTG CCTGCTTTGG 881 AGTCAATTCT GCAAGAGCAC AAGGGTAAGT ATGTGCGCCT 921 GATTGGGATT GACCCTGCGG CTCGTCGTCG CGTGGCTGAA 961 CTGTTGATTC AAAAGCCGGG ATCTCGCAAG CTCATCGAGG 1001 GGTTACGGCA TTTCCGTACG TCCTACTACC CGTCTCATCG 1041 GGACCTGTTC GAGCAGTTTG CCAAAGGTCA GCACCCTCGA 1081 GTCCTGTTCA TTACCTGCTC AGACTCGCGC ATTGACCCTA 1121 ACCTCATTAC CCAGTCGGGC ATGGGTGAGC TGTTCGTCAT 1161 TCGCAACGCT GGCAATCTGA TCCCGCCCTT CGGTGCCGCC 1201 AACGGTGGTG AAGGGGCATC GATCGAATAC GCGATCGCAG 1241 CTTTGAACAT TGAGCATGTT GTGGTCTGCG GTCACTCGCA 1281 CTGCGGTGCG ATGAAAGGGC TGCTCAAGCT CAATCAGCTG 1321 CAAGAGGACA TGCCGCTGGT CTATGACTGG CTGCAGCATG 1361 CCCAAGCCAC CCGCCGCCTA GTCTTGGATA ACTACAGCGG 1401 TTATGAGACT GACGACTTGG TAGAGATTCT GGTCGCCGAG 1441 AATGTGCTGA CGCAGATCGA GAACCTTAAG ACCTACCCGA 1481 TCGTGCGATC GCGCCTTTTC CAAGGCAAGC TGCAGATTTT 1521 TGGCTGGATT TATGAAGTTG AAAGCGGCGA GGTCTTGCAG 1561 ATTAGCCGTA CCAGCAGTGA TGACACAGGC ATTGATGAAT 1601 GTCCAGTGCG TTTGCCCGGC AGCCAGGAGA AAGCCATTCT 1641 CGGTCGTTGT GTCGTCCCCC TGACCGAAGA AGTGGCCGTT 1681 GCTCCACCAG AGCCGGAGCC TGTGATCGCG GCTGTGGCGG 1721 CTCCACCCGC CAACTACTCC AGTCGCGGTT GGTTGGGATC 1761 TGGAGGCAGT GTCTACGGCA AGGAACAGTT TTTGCGGATG 1801 CGCCAGAGCA TGTTCCCCGA TCGCTAA

Another nucleotide sequence is provided below that encodes the chimeric core carboxysome CcmC protein and that has been codon-optimized for expression in Escherichia coli (SEQ ID NO:27).

1 ATGACCGTTT CCGCGTACAA CGGACAGGGC AGACTTTCGA 41 GTGAAGTTAT AACCCAGGTC CGGTCTTTGT TGAACCAAGG 81 CTATCGCATC GGGACCGAAC ATGCCGATAA GCGCCGTTTC 121 CGGACCTCAA GTTGGCAACC GTGCGCGCCC ATCCAGTCAA 161 CCAATGAACG CCAGGTATTG TCTGAATTAG AGAATTGCTT 201 ATCGGAACAC GAAGGAGAAT ACGTTCGCTT GTTAGGAATT 241 GACACTAACA CAAGAAGTCG GGTTTTCGAA GCACTGATCC 281 AGCGCCCGGA CGGGTCTGTT CCTGAATCTT TGGGCAGCCA 321 GCCAGTAGCA GTGGCTTCCG GAGGCGGAAG ACAATCGTCC 361 TATGCATCTG TTTCCGGCAA CTTGTCTGCT GAGGTTGTTA 401 ATAAGGTGCG CAACCTGCTT GCCCAGGGTT ACAGAATTGG 441 CACGGAGCAC GCCGATAAGC GCCGTTTTAG AACCAGCTCG 481 TGGCAGTCTT GTGCGCCGAT ACAGTCCTCG AATGAACGGC 521 AGGTGCTGGC AGAGTTAGAG AATTGCCTGA GTGAGCATGA 561 AGGAGAATAC GTCCGCCTTC TGGGCATTGA CACCGCTTCC 601 CGTTCGCGTG TTTTCGAAGC CCTTATTCAG GATCCGCAAG 641 GCCCCGTGGG TTCCGCCAAA GCTGCCGCAG CACCTGTATC 681 AAGTGCTACC CCTTCGTCCC ACAGTTATAC GTCGAACGGC 721 AGCTCATCAT CTGACGTGGC GGGCCAGGTT CGTGGGTTGT 761 TGGCTCAAGG GTATCGGATA TCGGCTGAGG TTGCGGATAA 801 ACGTCGGTTC CAAACATCGT CGTGGCAGTC CTTGCCTGCA 841 TTATCGGGTC AATCGGAAGC AACGGTCCTT CCTGCGCTGG 881 AGAGTATCCT TCAGGAGCAC AAGGGCAAGT ACGTCAGATT 921 GATAGGGATC GATCCGGCGG CGCGGAGACG GGTGGCAGAA 961 TTGCTTATCC AAAAACCCGG TTCGCGCAAG TTGATCGAAG 1001 GATTAAGACA TTTTAGAACC TCATATTACC CGAGTCATAG 1041 AGATTTATTC GAGCAGTTTG CAAAGGGTCA ACACCCTAGA 1081 GTCCTGTTCA TCACTTGCTC GGATTCACGG ATCGATCCTA 1121 ATTTGATCAC GCAGTCTGGT ATGGGAGAGC TTTTCGTCAT 1161 CCGTAACGCA GGTAACCTGA TTCCACCTTT CGGCGCGGCA 1201 AATGGGGGTG AGGGTGCGTC CATTGAATAT GCCATCGCCG 1241 CATTGAATAT CGAACACGTA GTTGTATGTG GCCACTCGCA 1281 CTGTGGAGCG ATGAAAGGGC TGCTGAAGCT TAACCAGCTG 1321 CAAGAAGACA TGCCCCTTGT TTACGATTGG TTGCAACACG 1361 CGCAGGCCAC GAGACGTCTG GTCCTTGACA ACTACAGCGG 1401 ATATGAAACG GACGACCTTG TCGAGATCCT GGTCGCCGAG 1441 AACGTATTGA CCCAAATAGA GAATCTGAAG ACCTACCCAA 1481 TTGTGCGCTC GCGCTTGTTC CAGGGTAAGT TACAAATTTT 1521 CGGTTGGATC TATGAAGTGG AAAGTGGAGA GGTCTTGCAA 1561 ATCTCACGTA CATCCTCGGA CGACACAGGA ATAGACGAGT 1601 GCCCCGTCCG TTTACCGGGA TCGCAAGAGA AGGCCATTTT 1641 AGGACGGTGC GTCGTGCCAC TGACAGAGGA AGTGGCTGTT 1681 GCCCCTCCAG AACCAGAGCC TGTCATTGCT GCGGTGGCCG 1721 CACCACCCGC GAATTACTCC AGTCGCGGTT GGCTGGGCTC 1761 TGGAGGCTCT GTCTACGGAA AGGAACAATT CCTTCGTATG 1801 CGGCAATCAA TGTTCCCGGA CCGCTAA

Another nucleotide sequence is provided below that encodes the chimeric core carboxysome CcmC protein and that has been codon-optimized for expression in Nicotiana tabacum (SEQ ID NO:28).

1 ATGACTGTGA GTGCATATAA TGGACAAGGT AGATTGAGTT 41 CTGAAGTGAT AACTCAAGTG CGTAGCCTTT TGAATCAAGG 81 ATACAGAATT GGGACCGAAC ACGCAGATAA AAGAAGGTTT 121 AGAACCAGTT CATGGCAGCC ATGCGCCCCC ATCCAGTCTA 161 CTAATGAAAG ACAAGTGCTT TCTGAGCTGG AAAACTGTCT 201 TAGTGAACAT GAAGGCGAGT ATGTGCGATT GCTGGGTATC 241 GATACTAACA CTCGTAGCCG TGTTTTTGAA GCTCTGATAC 281 AACGACCTGA CGGTAGTGTC CCCGAATCAC TGGGTAGCCA 321 GCCCGTAGCA GTAGCTAGCG GGGGCGGGCG ACAGTCCTCC 361 TACGCCTCTG TTAGCGGCAA CCTCTCAGCC GAAGTAGTGA 401 ACAAAGTAAG AAACCTCCTC GCCCAGGGTT ACCGTATAGG 441 AACCGAGCAC GCAGACAAAC GAAGATTCAG GACTAGCAGC 481 TGGCAATCCT GCGCACCCAT ACAATCTTCC AACGAAAGAC 521 AGGTACTGGC AGAATTGGAA AACTGTCTTT CAGAACATGA 561 AGGCGAGTAC GTCCGTCTGC TGGGGATCGA CACAGCAAGC 601 AGAAGCCGAG TATTTGAAGC CCTCATTCAA GATCCACAGG 641 GGCCAGTAGG TAGTGCAAAG GCAGCTGCAG CTCCCGTTTC 681 ATCTGCTACT CCCAGCAGTC ACAGCTACAC TTCTAATGGG 721 TCTTCCAGTA GTGACGTCGC CGGACAGGTA AGAGGCCTGT 761 TGGCACAGGG TTACCGAATA TCTGCCGAAG TAGCTGATAA 801 AAGGCGATTC CAGACTTCAT CCTGGCAGTC CCTTCCTGCA 841 TTGTCTGGCC AATCTGAAGC CACTGTTCTT CCTGCACTTG 881 AATCCATTTT GCAGGAACAT AAAGGTAAGT ATGTTCGATT 921 GATCGGTATC GATCCAGCTG CACGTAGAAG GGTTGCAGAG 961 TTATTGATTC AGAAGCCAGG ATCTCGAAAA TTAATAGAGG 1001 GTTTACGACA TTTCAGAACT TCTTACTACC CTTCCCATCG 1041 TGACTTATTC GAGCAATTTG CAAAAGGCCA ACATCCCAGA 1081 GTCTTGTTTA TCACTTGTTC AGACTCTCGA ATAGACCCCA 1121 ATCTCATAAC ACAGTCTGGA ATGGGCGAGC TTTTCGTGAT 1161 ACGTAACGCC GGCAACCTCA TTCCTCCCTT TGGTGCAGCT 1201 AACGGGGGCG AGGGGGCTTC AATAGAGTAC GCTATCGCTG 1241 CCCTCAATAT CGAACACGTC GTAGTATGCG GACATTCACA 1281 TTGCGGGGCC ATGAAGGGAC TGTTGAAGCT GAATCAACTC 1321 CAAGAGGACA TGCCCCTGGT CTATGATTGG TTGCAGCACG 1361 CCCAAGCTAC TAGGAGATTA GTTTTAGACA ACTACTCTGG 1401 CTATGAAACT GATGACCTGG TAGAAATACT GGTCGCAGAA 1441 AACGTATTAA CTCAGATAGA AAATTTAAAG ACTTATCCCA 1481 TAGTCCGTAG CCGATTGTTC CAAGGAAAAT TGCAAATATT 1521 CGGGTGGATC TATGAGGTTG AGTCCGGAGA GGTCTTGCAG 1561 ATAAGTCGAA CTAGCTCCGA CGACACAGGG ATAGACGAAT 1601 GCCCAGTCAG GTTGCCCGGG TCTCAAGAGA AAGCTATCTT 1641 GGGGAGGTGT GTCGTTCCTT TAACCGAGGA AGTTGCTGTC 1681 GCCCCCCCTG AGCCTGAACC TGTGATAGCT GCCGTAGCCG 1721 CACCCCCTGC CAACTATTCA TCACGAGGCT GGCTTGGCTC 1761 AGGGGGCTCA GTTTATGGGA AGGAACAATT CCTGAGGATG 1801 AGACAGTCAA TGTTCCCCGA TAGATAA

Another nucleotide sequence is provided below that encodes the chimeric core carboxysome CcmC protein and that has been codon-optimized for expression in Chlamydomonas reinhardtii (SEQ ID NO:29).

1 ATGACGGTGT CGGCTTACAA CGGCCAGGGC CGCCTCTCGT 41 CCGAGGTCAT TACGCAGGTC CGGAGCCTCC TGAACCAGGG 81 GTACCGGATT GGTACCGAGC ATGCCGACAA GCGGCGCTTT 121 CGGACGTCGT CCTGGCAGCC CTGCGCGCCC ATTCAGAGCA 161 CCAACGAGCG GCAGGTCCTC TCCGAGCTGG AGAACTGCCT 201 CAGCGAGCAT GAGGGGGAGT ACGTCCGCCT GCTGGGGATC 241 GATACGAACA CGCGCTCCCG GGTCTTCGAG GCTCTCATCC 281 AGCGCCCTGA CGGCTCGGTG CCTGAGAGCC TCGGCTCGCA 321 GCCTGTGGCC GTGGCGAGCG GCGGTGGGCG GCAGTCCAGC 361 TACGCCAGCG TGTCGGGTAA CCTCTCCGCC GAGGTCGTCA 401 ACAAGGTGCG GAACCTCCTG GCCCAGGGCT ACCGGATCGG 441 TACCGAGCAC GCCGACAAGC GCCGCTTTCG CACGAGCTCG 481 TGGCAGAGCT GCGCCCCCAT TCAGTCGAGC AACGAGCGGC 521 AGGTGCTCGC TGAGCTGGAG AACTGCCTCT CCGAGCATGA 561 GGGCGAGTAC GTGCGGCTGC TCGGGATTGA TACGGCCTCG 601 CGGTCGCGCG TGTTTGAGGC GCTGATCCAG GACCCCCAGG 641 GTCCTGTCGG TTCGGCTAAG GCTGCGGCTG CCCCTGTGTC 681 CTCGGCCACC CCCAGCTCGC ATTCGTACAC CTCGAACGGC 721 TCCTCGTCGT CCGATGTGGC GGGTCAGGTG CGCGGGCTCC 761 TCGCTCAGGG CTACCGCATC AGCGCTGAGG TCGCCGATAA 801 GCGGCGGTTT CAGACGAGCT CGTGGCAGTC CCTCCCGGCG 841 CTCTCGGGTC AGAGCGAGGC CACCGTCCTC CCTGCTCTCG 881 AGTCGATTCT CCAGGAGCAT AAGGGGAAGT ACGTCCGGCT 921 CATCGGGATT GACCCGGCTG CTCGGCGCCG CGTGGCGGAG 961 CTGCTGATTC AGAAGCCTGG CAGCCGGAAG CTCATCGAGG 1001 GGCTCCGCCA TTTCCGGACG TCCTACTACC CCTCCCACCG 1041 CGATCTCTTC GAGCAGTTTG CCAAGGGGCA GCACCCGCGG 1081 GTCCTGTTCA TTACGTGCTC CGATAGCCGC ATTGACCCGA 1121 ACCTCATCAC GCAGAGCGGT ATGGGTGAGC TCTTTGTGAT 1161 TCGCAACGCT GGTAACCTCA TTCCTCCCTT TGGGGCGGCG 1201 AACGGCGGCG AGGGTGCGTC GATTGAGTAC GCTATCGCCG 1241 CCCTCAACAT TGAGCATGTC GTGGTGTGCG GTCATAGCCA 1281 TTGCGGCGCG ATGAAGGGCC TCCTCAAGCT GAACCAGCTG 1321 CAGGAGGACA TGCCTCTGGT GTACGACTGG CTGCAGCATG 1361 CTCAGGCTAC GCGGCGCCTC GTCCTGGACA ACTACTCGGG 1401 CTACGAGACC GATGACCTCG TCGAGATCCT CGTCGCGGAG 1441 AACGTGCTGA CCCAGATTGA GAACCTCAAG ACGTACCCCA 1481 TCGTGCGCTC GCGCCTCTTC CAGGGCAAGC TGCAGATCTT 1521 CGGTTGGATT TACGAGGTGG AGTCGGGGGA GGTCCTGCAG 1561 ATCAGCCGGA CGAGCTCCGA CGACACCGGG ATCGATGAGT 1601 GCCCTGTCCG CCTGCCGGGC TCGCAGGAGA AGGCCATTCT 1641 GGGTCGGTGC GTGGTCCCCC TGACGGAGGA GGTGGCTGTG 1681 GCTCCTCCCG AGCCTGAGCC CGTCATTGCG GCGGTCGCCG 1721 CCCCTCCGGC TAACTACTCC AGCCGGGGGT GGCTCGGCTC 1761 CGGGGGGAGC GTCTACGGCA AGGAGCAGTT TCTGCGCATG 1801 CGGCAGTCGA TGTTCCCGGA TCGCTAA

The expression cassettes or vectors can include a promoter that is operably linked to a nucleic acid segment that encodes the chimeric core carboxysome CcmC protein. A promoter is a nucleotide sequence that controls expression of an operably linked nucleic acid sequence by providing a recognition site for RNA polymerase, and possibly other factors, required for proper transcription. A promoter includes a minimal promoter, consisting only of all basal elements needed for transcription initiation, such as a TATA-box and/or other sequences that serve to specify the site of transcription initiation. A promoter may be obtained from a variety of different sources. For example, a promoter may be derived entirely from a native gene, be composed of different elements derived from different promoters found in nature, or be composed of nucleic acid sequences that are entirely synthetic. A promoter may be derived from many different types of organisms and tailored for use within a given cell.

Any promoter able to direct transcription of an encoded peptide or polypeptide may be used. Accordingly, many promoters may be included within the expression cassette. Some useful promoters include constitutive promoters, inducible promoters, regulated promoters, cell specific promoters, viral promoters, and synthetic promoters. Particularly useful promoters are inducible promoters, especially those induced by inexpensive signals, or promoters that are auto-inducing under certain environmental conditions (e.g. a relatively dense cyanobacterial population).

For expression of one or more chimeric carboxysome core protein, shell protein, Rubisco protein, or combinations thereof in a host cell, one or more expression cassette can be used that has a nucleic acid segment encoding such protein(s) and a promoter operably linked thereto. Such a promoter can be any DNA sequence capable of binding a RNA polymerase and initiating the downstream (3″) transcription of a coding sequence into mRNA. A promoter has a transcription initiation region that is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. A second domain called an operator may be present and overlap an adjacent RNA polymerase binding site at which RNA synthesis begins. The operator permits negatively regulated (inducible) transcription, as a gene repressor protein may bind the operator and thereby inhibit transcription of a specific gene.

Constitutive expression may occur in the absence of negative regulatory elements, such as the operator. In addition, positive regulation may be achieved by a gene activator protein binding sequence, which, if present is usually proximal (5′) to the RNA polymerase binding sequence. An example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in E. coli (Raibaud et al., Ann. Rev. Genet., 18:173 (1984)). Regulated expression may therefore be positive or negative, thereby either enhancing or reducing transcription.

Other examples of promoters that can be employed include promoters of sugar metabolizing enzymes, such as galactose, lactose (lac) (Chang et al., Nature, 198:1056 (1977), and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (Trp) (Goeddel et al., Nuc. Acids Res., 8:4057 (1980); Yelverton et al., Nuc. Acids Res., 9:731 (1981); U.S. Pat. No. 4,738,921; and EPO Publ. Nos. 036 776 and 121 775). The β-lactamase (bla) promoter system (Weissmann, “The cloning of interferon and other mistakes”, in: Interferon 3 (ed. I. Gresser), 1981), and bacteriophage lambda P_(L) (Shimatake et al., Nature, 292:128 (1981)) and T5 (U.S. Pat. No. 4,689,406) promoter systems also provide useful promoter sequences. Another example is the Chlorella virus promoter (U.S. Pat. No. 6,316,224).

Synthetic promoters that do not occur in nature also function as promoters in host cells. For example, transcription activation sequences of a promoter may be joined with the operon sequences of another promoter, creating a synthetic hybrid promoter (U.S. Pat. No. 4,551,433). For example, the tac promoter is a hybrid trp-lac promoter comprised of both trp promoter and lac operon sequences that is regulated by the lac repressor (Amann et al., Gene, 25:167 (1983); de Boer et al., Proc. Natl. Acad. Sci. USA, 80:21 (1983)). Furthermore, a promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind RNA polymerase and initiate transcription in cyanobacteria or other types of host cells. A naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes. The bacteriophage T7 RNA polymerase/promoter system is an example of a coupled promoter system (Studier et al., J. Mol. Biol., 189:113 (1986); Tabor et al., Proc. Natl. Acad. Sci. USA, 82:1074 (1985)). In addition, a hybrid promoter can also be comprised of a bacteriophage promoter and an E. coli operator region (EPO Publ. No. 267 851).

In some cases, quorum sensing-responsive promoters can be employed in the expression cassettes/vectors. Quorum sensing is a mechanism whereby bacteria are able to indirectly detect the concentration of neighboring cells. A quorum sensing pathway is one that is usually activated when a bacterial population becomes concentrated. For example, biofilm formation is controlled often by quorum sensing. Such quorum sensing promoters can make bacteria, cyanobacteria, or other cells self-induce the genes of interest when a certain cell concentration is reached (e.g., when the cells are ready, or will soon be ready, to be harvested), without the addition of chemical inducers. See, e.g., Miller, Melissa B., and Bonnie L. Bassler, “Quorum sensing in bacteria.” Annual Reviews in Microbiology 55(1): 165-199 (2001).

In some cases, the promoter can become active at certain times during culture or fermentation. For example, the promoter can in some cases be active before, during, or after log phase growth of the cells during culture or fermentation.

For example, LuxI/LuxR genes are a family of genes that produce quorum sensing behavior in bacteria. See, e.g., Waters & Bassler, “Quorum sensing: cell-to-cell communication in bacteria,” Ann Rev Cell Dev Biol 21: 319-46 (2005). Quorum sensing pathways in natural contexts involve a microbe that is capable of producing a diffusible molecule that can pass through the cell membrane, such as the class of molecules called acyl-homoserine lactones (AHL). These molecules can diffuse from the cell that produces them to the outside environment, and then back into other neighboring bacteria. When the concentration of AHL of a specific type becomes high enough, it can stabilize a transcription factor that turns on specific genes. Usually, quorum sensing pathways are utilized for bacteria to sense how large its population is—the more surrounding bacteria in the environment, the higher the AHL levels. At a certain cell density, the AHL builds up to a level that it can bind a receptor protein (e.g. LuxR), stabilizing it and allowing for downstream gene regulation.

Quorum sensing-responsive promoters can be used in any of the expression cassettes or expression vectors described herein. For example, host cells expressing LuxI (or similar protein) can make an AHL signal that could then build up as the cell density increases. When the cells become dense enough, they can turn on the expression of chimeric carboxysome core protein(s), shell protein(s), Rubisco protein(s), or combinations thereof.

One example of a protein that can modulate quorum sensing-responsive promoters is the LuxI from Vibrio fishcheri, with the following sequence (SEQ ID NO:30).

1 MIKKSDFLGI PSEEYRGILS LRYQVFKRRL EWDLVSEDNL 41 ESDEYDNSNA EYIYACDDAE EVNGCWRLLP TTGDYMLKTV 81 FPELLGDQVA PRDPNIVELS RFAVGKNSSK INNSASEITM 121 KLFQAIYKHA VSQGITEYVT VTSIAIERFL KRIKVPCHRI 161 GDKEIHLLGN TRSVVLSMPI NDQFRKAVSN A nucleic acid encoding this Vibrio fishcheri LuxI protein shown below (SEQ ID NO:31).

1 ATGATAAAAA AATCGGACTT TTTGGGCATT CCATCAGAGG 41 AGTATAGAGG TATTCTTAGT CTTCGTTATC AGGTATTTAA 81 ACGAAGACTG GAGTGGGACT TGGTAAGTGA GGATAATCTT 121 GAATCAGATG AATATGATAA CTCAAATGCA GAATATATTT 161 ATGCTTGTGA TGATGCGGAA GAGGTAAATG GCTGTTGGCG 201 TTTGTTACCT ACAACGGGTG ATTACATGTT AAAAACTGTT 241 TTTCCTGAAT TGCTCGGAGA TCAAGTAGCC CCAAGAGATC 281 CAAATATAGT CGAATTAAGC CGTTTTGCTG TGGGAAAAAA 321 TAGCTCAAAA ATAAATAACT CTGCTAGTGA AATAACAATG 361 AAATTGTTTC AAGCTATATA TAAACACGCA GTTAGTCAAG 401 GTATTACAGA ATATGTAACA GTAACATCAA TAGCAATAGA 441 GCGATTTCTG AAACGTATTA AAGTTCCTTG TCATCGCATT 481 GGTGATAAGG AGATTCATTT ATTAGGTAAT ACTAGATCTG 521 TTGTATTGTC TATGCCTATT AATGATCAGT TTAGAAAAGC 561 TGTATCAAAT TAA

A sequence of a LuxR receptor protein from Vibrio fishcheri is shown below (SEQ ID NO:32).

1 MIYNTQNLRQ TIGKDKEMGM KNINADDTYR IINKIKACRS 41 NNDINQCLSD MTKMVHCEYY LLAIIYPHSM VKSDISILDN 81 YPKKWRQYYD DANLIKYDPI VDYSNSNHSP INWNIFENNA 121 VNKKSPNVIK EAKTSGLITG FSFPIHTANN GFGMLSFAHS 161 EKDNYIDSLF LHACMNIPLI VPSLVDNYRK INIANNKSNN 201 DLTKREKECL AWACEGKSSW DISKILGCSE RTVTFHLTNA 241 QMKLNTTNRC QSISKAILTG AIDCPYFKN A nucleic acid sequence for this LuxR protein from Vibrio fishcheri is provided below as SEQ ID NO:33.

1 ATGATATATA ACACGCAAAA CTTGCGACAA ACAATAGGTA 41 AGGATAAAGA GATGGGTATG AAAAACATAA ATGCCGACGA 81 CACATACAGA ATAATTAATA AAATTAAAGC TTGTAGAAGC 121 AATAATGATA TTAATCAATG CTTATCTGAT ATGACTAAAA 161 TGGTACATTG TGAATATTAT TTACTCGCGA TCATTTATCC 201 TCATTCTATG GTTAAATCTG ATATTTCAAT TCTAGATAAT 241 TACCCTAAAA AATGGAGGCA ATATTATGAT GACGCTAATT 281 TAATAAAATA TGATCCTATA GTAGATTATT CTAACTCCAA 321 TCATTCACCA ATTAATTGGA ATATATTTGA AAACAATGCT 361 GTAAATAAAA AATCTCCAAA TGTAATTAAA GAAGCGAAAA 401 CATCAGGTCT TATCACTGGG TTTAGTTTCC CTATTCATAC 441 GGCTAACAAT GGCTTCGGAA TGCTTAGTTT TGCACATTCA 481 GAAAAAGACA ACTATATAGA TAGTTTATTT TTACATGCGT 521 GTATGAACAT ACCATTAATT GTTCCTTCTC TAGTTGATAA 561 TTATCGAAAA ATAAATATAG CAAATAATAA ATCAAACAAC 601 GATTTAACCA AAAGAGAAAA AGAATGTTTA GCGTGGGCAT 641 GCGAAGGAAA AAGCTCTTGG GATATTTCAA AAATATTAGG 681 CTGCAGTGAG CGTACTGTCA CTTTCCATTT AACCAATGCG 721 CAAATGAAAC TCAATACAAC AAACCGCTGC CAAAGTATTT 761 CTAAAGCAAT TTTAACAGGA GCAATTGATT GCCCATACTT 801 TAAAAATTAA

An example of a LuxR-responsive promoter from Vibrio fishcheri is shown below as (SEQ ID NO:34).

1 TGTCGCAAGT TTTGCGTGTT ATATATCATT AAAACGGTAA 41 TGGATTGACA TTTGATTCTA ATAAATTGGA TTTTTGTCAC 81 ACTATTGTAT CGCTGGGAAT ACAATTACTT AACATAAGCA 121 CCTGTAGGAT CGTACAGGTT TACGCAAGAA AATGGTTTGT 161 TATAGTCGAA TGAATTCATT AAAGAGGAGA AAGGTACC When LuxR is expressed and stabilized (because AHL is present), the LuxR protein binds to a promoter sequence like that shown above as (SEQ ID NO:34) and drives gene expression from it.

It is understood that many promoters and associated regulatory elements may be used within the expression cassette/vector to transcribe an RNA encoding a chimeric carboxysome core protein. The promoters described above are provided merely as examples and are not to be considered as a complete list of promoters that are included within the scope of the invention.

The expression cassette of the invention may contain a nucleic acid sequence for increasing the translation efficiency of an mRNA encoding a chimeric carboxysome core protein. Such increased translation serves to increase production of the protein. The presence of an efficient ribosome binding site is useful for gene expression in prokaryotes. In bacterial mRNA, a conserved stretch of six nucleotides, the Shine-Dalgarno sequence, is usually found upstream of the initiating AUG codon. (Shine et al., Nature, 254:34 (1975)). This sequence is thought to promote ribosome binding to the mRNA by base pairing between the ribosome binding site and the 3′ end of Escherichia coli 16S rRNA. (Steitz et al., “Genetic signals and nucleotide sequences in messenger RNA”, in: Biological Regulation and Development: Gene Expression (ed. R. F. Goldberger), 1979)). Such a ribosome binding site, or operable derivatives thereof, are included within the expression cassette of the invention.

A translation initiation sequence can be derived from any expressed gene and can be used within an expression cassette/vector of the invention. Preferably the gene from which the translation initiation sequence is obtained is a highly expressed gene. A translation initiation sequence can be obtained via standard recombinant methods, synthetic techniques, purification techniques, or combinations thereof, which are all well known. (Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY. (1989); Beaucage and Caruthers, Tetra. Letts., 22:1859 (1981); VanDevanter et al., Nucleic Acids Res., 12:6159 (1984). Alternatively, translational start sequences can be obtained from numerous commercial vendors. (Operon Technologies; Life Technologies Inc, Gaithersburg, Md.). In some embodiments, the T7 translation initiation sequence is used. The T7 translation initiation sequence is derived from the highly expressed T7 Gene 10 cistron and can have a sequence that includes TCTAGAAATAATTTTGTTTAACTTTAAGAA GGAGATATA (SEQ ID NO:35). Other examples of translation initiation sequences include, but are not limited to, the maltose-binding protein (Mal E gene) start sequence (Guan et al., Gene, 67:21 (1997)) present in the pMalc2 expression vector (New England Biolabs, Beverly, Mass.) and the translation initiation sequence for the following genes: thioredoxin gene (Novagen, Madison, Wis.), Glutathione-S-transferase gene (Pharmacia, Piscataway, N.J.), β-galactosidase gene, chloramphenicol acetyltransferase gene and E. coli Trp E gene (Ausubel et al., 1989, Current Protocols in Molecular Biology, Chapter 16, Green Publishing Associates and Wiley Interscience, NY).

The invention therefore provides an expression cassette or vector that includes a promoter operable in a selected host and a nucleic acid encoding one of the chimeric carboxysome core proteins described herein. The expression cassette can have other elements, for example, termination signals, origins of replication, enhancers, and the like as described herein. The expression cassette can also be placed in a vector for easy replication and maintenance.

An expression cassette or nucleic acid construct of the invention is thought to be particularly advantageous for inducing expression of the polypeptides.

Host Organisms

The chimeric carboxysome core protein can be expressed by a variety of organisms. Examples of organisms that can be modified to express the chimeric carboxysome core protein can include microorganisms, plants (including land-based plants and aqueous plants), and fungi. For example, bacteria, cyanobacteria, algae, microalgae, seaweed, plankton, single-celled fungal cells, multi-celled fungi, plant cells, and multi-celled plants can be modified to express the chimeric carboxysome core protein.

In some cases, the chimeric carboxysome core protein can be expressed in addition to native or endogenous carboxysome components.

Any cyanobacteria can be modified to express the chimeric carboxysome core protein, either permanently or transiently.

Examples of cyanobacterial species that can be changed include Synechococcus elongatus sp. PCC 7942; Synechococcus elongatus 7002; Synechococcus elongatus UTEX 2973; Anthropira platensis; and Leptolyngbya sp. strain BL0902. Synechococcus elongatus sp. PCC 7942 is one of the dominant model organisms, providing a variety of useful genetic tools. Synechococcus elongatus 7002 is a well-developed model organism with improved productivity and resilience. Synechococcus elongatus UTEX 2973 is related to S. elongatus 7942, and it has greatly improved growth properties. Anthropira platensis is perhaps the most broadly utilized cyanobacteria in scaled applications. Leptolyngbya sp. strain BL0902 is a bioindustrial strain whose genetic make-up is not as well-studied as some of the model cyanobacterial species.

Further examples of cyanobacterial species that can be modified include, for example, any of those in Table 1.

TABLE 1 Types of Cyanobacteria Species Lineage Release Synechococcus Cyanobacteria; Oscillatorio- American Type Culture elongatus sp. PCC 7942 phycideae; Chroococcales; Collection, ATCC Synechococcus accession no. 33912. Synechococcus Cyanobacteria; Oscillatorio- UTEX Culture Collection elongatus UTEX 2973 phycideae; Chroococcales; of Algae, University of Synechococcus Texas at Austin Anthropira platensis Cyanobacteria; Oscillatorio- American Type Culture phycideae; Oscillatoriales; Collection, ATCC Arthrospira accession no. 29408. Prochlorococcus Cyanobacteria; Prochlorales; The Gordon and Betty marinus str. AS9601 Prochlorococcaceae; Moore Foundation Marine Prochlorococcus Microbiology Initiative (2007) Acaryochloris marina Cyanobacteria; Acaryochloris TGen Sequencing Center MBIC11017 (2008) Anabaena sp. PCC 7120 Cyanobacteria; Nostocales; Kazusa (2001) Nostocaceae; Nostoc Anabaena variabilis Cyanobacteria; Nostocales; JGI (2007) ATCC 29413 Nostocaceae; Anabaena Synechococcus sp. Cyanobacteria; Chroococcales; TIGR (2006) CC9311 Synechococcus Cyanothece sp. ATCC Cyanobacteria; Chroococcales; Washington University 51142 Cyanothece (2008) Chlorobium tepidum Chlorobi; Chlorobia; TIGR (2002) TLS Chlorobiales; Chlorobiaceae; Chlorobaculum Synechococcus sp. JA-3- Cyanobacteria; Chroococcales; TIGR (2007) 3Ab Synechococcus Cyanothece sp. PCC Cyanobacteria; Chroococcales; 7425 Cyanothece Synechococcus sp. JA-2- Cyanobacteria; Chroococcales; TIGR (2007) 3B′a(2-13) Synechococcus Gloeobacter violaceus Cyanobacteria; Gloeobacteria; Kazusa (2003) PCC 7421 Gloeobacterales; Gloeobacter Prochlorococcus Cyanobacteria; Prochlorales; JGI (2003) marinus MED4 Prochlorococcaceae; Prochlorococcus Microcystis aeruginosa Cyanobacteria; Chroococcales; Kazusa, Tsukuba, NIES NIES-843 Microcystis (2007) Prochlorococcus Cyanobacteria; Prochlorales; JGI (2003) marinus MIT9313 Prochlorococcaceae; Prochlorococcus Prochlorococcus Cyanobacteria; Prochlorales; The Gordon and Betty marinus str. NATL1A Prochlorococcaceae; Moore Foundation Marine Prochlorococcus Microbiology Initiative (2007) Arthrospira platensis Cyanobacteria; Oscillatoriales; NIES-39 Arthrospira; Arthrospira platensis Nostoc punctiforme Cyanobacteria; Nostocales; JGI (2008) ATCC 29133 Nostocaceae; Nostoc Prochlorococcus Cyanobacteria; Prochlorales; The Gordon and Betty marinus str. MIT 9211 Prochlorococcaceae; Moore Foundation Marine Prochlorococcus Microbiology Initiative (2008) Prochlorococcus Cyanobacteria; Prochlorales; JGI (2007) marinus str. MIT 9215 Prochlorococcaceae; Prochlorococcus Prochlorococcus Cyanobacteria; Prochlorales; The Gordon and Betty marinus str. MIT 9301 Prochlorococcaceae; Moore Foundation Marine Prochlorococcus Microbiology Initiative (2007) Prochlorococcus Cyanobacteria; Prochlorales; The Gordon and Betty marinus str. MIT 9303 Prochlorococcaceae; Moore Foundation Marine Prochlorococcus Microbiology Initiative (2007) Prochlorococcus Cyanobacteria; Prochlorales; The Gordon and Betty marinus str. MIT 9515 Prochlorococcaceae; Moore Foundation Marine Prochlorococcus Microbiology Initiative (2007) Synechococcus Cyanobacteria; Chroococcales; Nagoya U. (2007) elongatus PCC 6301 Synechococcus Cyanothece sp. PCC Cyanobacteria; Chroococcales; 7424 Cyanothece Cyanothece sp. PCC Cyanobacteria; Chroococcales; 8801 Cyanothece Prochlorococcus Cyanobacteria; Prochlorales; JGI (2007) marinus str. NATL2A Prochlorococcaceae; Prochlorococcus Prochlorococcus Cyanobacteria; Prochlorales; JGI (2007) marinus str. MIT 9312 Prochlorococcaceae; Prochlorococcus Rhodopseudomonas Proteobacteria; JGI (2003) palustris CGA009 Alphaproteobacteria; Rhizobiales; Bradyrhizobiaceae; Rhodopseudomonas Prochlorococcus Cyanobacteria; Prochlorales; Genoscope (2003) marinus SS120 Prochlorococcaceae; Prochlorococcus Synechococcus sp. Cyanobacteria; Chroococcales; JGI (2007) CC9605 Synechococcus Synechococcus sp. Cyanobacteria; Chroococcales; JGI (2007) CC9902 Synechococcus Synechocystis sp. PCC Cyanobacteria; Chroococcales; Kazusa (1996, 2002, 2003) 6803 Synechocystis Synechococcus sp. PCC Cyanobacteria; Chroococcales; Penn State University 7002 Synechococcus (2008) Synechococcus Cyanobacteria; Chroococcales; JGI (2007) elongatus PCC 7942 Synechococcus Synechococcus sp. Cyanobacteria; Chroococcales; Genoscope (2007) RCC307 Synechococcus Synechococcus sp. WH Cyanobacteria; Chroococcales; Genoscope (2007) 7803 Synechococcus Trichodesmium Cyanobacteria; Oscillatoriales; erythraeum IMS101 Trichodesmium; Trichodesmium erythraeum Thermosynechococcus Cyanobacteria; Chroococcales; Kazusa (2002) elongatus BP-1 Thermosynechococcus Synechococcus sp. Cyanobacteria; Chroococcales; JGI (2003) WH8102 Synechococcus Useful Products

The cells, plants, cyanobacteria, bacteria, algae, microalgae and other cells/organisms that express the fusion proteins described herein can produce a variety of products such as oils, carbohydrates, grains, vegetables, fruits and other components, as well as 3-phosphoglycerate (3-PGA). Examples include oils (fatty acids), alkenes, polyhydroxybutyrate, biomass, carbohydrates, phycocyanin, ethanol, hydrogen, isobutanol, ethylene, and combinations thereof. Products such as oils (fatty acids), alkenes, ethanol, hydrogen, isobutanol, ethylene, and combinations thereof can be used in manufacturing and as biofuels. For example, ethanol, carbohydrate feedstocks, and biomass can be used to make bioethanol. Polyhydroxybutyrate is useful, for example, in bioplastics. Biomass, carbohydrates, and ethanol can also be used in foods and food manufacturing. Ethanol, hydrogen, isobutanol, and ethylene are useful in manufacturing, as a source of energy, and/or for making fuel.

The following non-limiting Examples describe some of the experiments performed.

Example 1: Materials and Methods

This Example describes some of the methods that were used during development of the invention.

Cyanobacterial Strain and Growth Conditions

Synechococcus elongatus PCC 7942 (Syn 7942) cultures were grown in 250 ml baffled Erlenmeyer flasks with 60 ml BG-11 medium (Rippka et al., 1979) buffered with 10 mM HEPES pH 8.0 under the following growth chamber settings: temperature of 30° C., light intensity of 40 moles photons m⁻²s⁻¹, shaking at 150 rpm and CO₂ concentrations of 5%, 3% or air. Unless otherwise indicated, experiments were performed in cultures at exponential growth phase (OD₇₃₀=0.4-0.7).

Mutant Generation

Synechococcus elongatus PCC 7942 cells were transformed as described by Kufryk et al. (2002). Cultures were grown to OD₇₃₀=0.5 and concentrated to OD₇₃₀=2.5 by centrifugation at 5000 relative centrifugal force (rcf) for 5 minutes. Five microliters of plasmids (˜1 μg of DNA) prepared from E. coli DH5α cells were added to 400 μl of the cyanobacterial cell suspension and incubated for 6 hours. The 400 μl-aliquots were dried on Nucleopore track-etched polycarbonate membranes (GE Healthcare) on top of BG-11 plates and incubated for 12-24 hours. The membranes were transferred to BG-11 plates with the proper selectable marker until resistant colonies were obtained.

All mutant strains were transformed with pJCC008 plasmid (rbcL-GFP placed under the control of the ccmk2 promoter) (Cameron et al., 2013) for GFP-labeling of the large subunit of Rubisco (RbcL) to enable carboxysome visualization by fluorescence microscopy. The carboxysome-minus strain COREΔ2/RbcL-GFP was generated by replacing synpcc7942_1423 and synpcc7942_1424 genes with a kanamycin resistance/sucrose sensitivity cassette obtained from the pPSBAII-KS plasmid (Lagarde et al., 2000) and using synpcc7942_1422 and synpcc7942_1425 sequences as flanking regions for double homologous recombination. Domains for the generation of chimeric proteins were assigned using the InterPro software (Hunter et al., 2012) and the HMM tool from JCVI institute (see website at blast.jcvi.org/web-hmm).

DNA was obtained from Cyanobase (see website at genome.microbedb.jp/cyanobase) and cloned by methods involving restriction digestion and ligation (see, e.g., Sambrook and Russell, 2001) as follows.

Plasmids with genes coding for the chimeric proteins had the following amino acid sequences.

The following is an amino acid sequence for a CcaA-M35 gene (SEQ ID NO:36).

1 MRKLIEGLRH FRTSYYPSHR DLFEQFAKGQ HPRVLFITCS 41 DSRIDPNLIT QSGMGELFVI RNAGNLIPPF GAANGGEGAS 81 IEYAIAALNI EHVVVCGHSH CGAMKGLLKL NQLQEDMPLV 121 YDWLQHAQAT RRLVLDNYSG YETDDLVEIL VAENVLTQIE 141 NLKTYPIVRS RLFQGKLQIF GWIYEVESGE VLQISRTSSD 181 DTGIDECPVR LPGSQEKAIL GRCVVPLTEE VAVAPPEPEP 221 VIAAVAAPPA NYSSRGWLAP EQQQRIYRGN ASGSVSAYNG 261 QGRLSSEVIT QVRSLLNQGY RIGTEHADKR RFRTSSWQPC 281 APIQSTNERQ VLSELENCLS EHEGEYVRLL GIDTNTRSRV 321 FEALIQRPDG SVPESLGSQP VAVASGGGRQ SSYASVSGNL 361 SAEVVNKVRN LLAQGYRIGT EHADKRRFRT SSWQSCAPIQ 401 SSNERQVLAE LENCLSEHEG EYVRLLGIDT ASRSRVFEAL 441 IQDPQGPVGS AKAAAAPVSS ATPSSHSYTS NGSSSSDVAG 481 QVRGLLAQGY RISAEVADKR RFQTSSWQSL PALSGQSEAT 521 VLPALESILQ EHKGKYVRLI GIDPAARRRV AELLIQKP

The following is an amino acid sequence for an M35-EP (SEQ ID NO:37).

1 MTVSAYNGQG RLSSEVITQV RSLLNQGYRI GTEHADKRRF 41 RTSSWQPCAP IQSTNERQVL SELENCLSEH EGEYVRLLGI 81 DTNTRSRVFE ALIQRPDGSV PESLGSQPVA VASGGGRQSS 121 YASVSGNLSA EVVNKVRNLL AQGYRIGTEH ADKRRFRTSS 161 WQSCAPIQSS NERQVLAELE NCLSEHEGEY VRLLGIDTAS 201 RSRVFEALIQ DPQGPVGSAK AAAAPVSSAT PSSHSYTSNG 241 SSSSDVAGQV RGLLAQGYRI SAEVADKRRF QTSSWQSLPA 281 LSGQSEATVL PALESILQEH KGKYVRLIGI DPAARRRVAE 321 LLIQKPGSGG SVYGKEQFLR MRQSMFPDR

The following is an amino acid sequence for a CcmC protein (SEQ ID NO:38).

1 MTVSAYNGQG RLSSEVITQV RSLLNQGYRI GTEHADKRRF 41 RTSSWQPCAP IQSTNERQVL SELENCLSEH EGEYVRLLGI 81 DTNTRSRVFE ALIQRPDGSV PESLGSQPVA VASGGGRQSS 121 YASVSGNLSA EVVNKVRNLL AQGYRIGTEH ADKRRFRTSS 161 WQSCAPIQSS NERQVLAELE NCLSEHEGEY VRLLGIDTAS 201 RSRVFEALIQ DPQGPVGSAK AAAAPVSSAT PSSHSYTSNG 241 SSSSDVAGQV RGLLAQGYRI SAEVADKRRF QTSSWQSLPA 281 LSGQSEATVL PALESILQEH KGKYVRLIGI DPAARRRVAE 321 LLIQKPGSRK LIEGLRHFRT SYYPSHRDLF EQFAKGQHPR 361 VLFITCSDSR IDPNLITQSG MGELFVIRNA GNLIPPFGAA 401 NGGEGASIEY AIAALNIEHV VVCGHSHCGA MKGLLKLNQL 441 QEDMPLVYDW LQHAQATRRL VLDNYSGYET DDLVEILVAE 481 NVLTQIENLK TYPIVRSRLF QGKLQIFGWI YEVESGEVLQ 521 ISRTSSDDTG IDECPVRLPG SQEKAILGRC VVPLTEEVAV 561 APPEPEPVIA AVAAPPANYS SRGWLGSGGS VYGKEQFLRM 601 RQSMFPDR Note that amino acids 1-328 of the CcmC protein (with SEQ ID NO:38) are the same as amino acids 1-328 of the M35-EP protein with SEQ ID NO:37. The central amino acids 329-585 (in bold) of the SEQ ID NO:38 CcmC protein correspond to amino acids 2-258 of the carbonate dehydratase (CcaA) with SEQ ID NO:71. Amino acids 591-608 of the SEQ ID NO:38 CcmC protein correspond to the encapsulation peptide from a CcmN protein, which has SEQ ID NO:13.

Note also that in the case of CcmC, the C-terminal extension of the β-CA was used as linker and its terminal 14 amino acids were replaced by 18 amino acids comprising the EP with synpcc7942_1422 and synpcc7942_1425 sequences as flanking regions were transformed into the COREΔ2/RbcL-GFP strain.

Growth in air was used for positive selection and growth in 5% sucrose as confirmation. The COREΔ2/CcmC/RbcL-GFP strain is obtained after CcmC restores growth in air. CcaA (Synpcc7942_1447) was interrupted in the COREΔ2/CcmC/RbcL-GFP strain and in Wild-type/RbcL-GFP by insertion of a gentamycin resistance cassette and selection with 5 μg/ml gentamycin in solid BG-11 plates (resulting in COREΔ3/CcmC/RbcL-GFP strain and ΔCcaA/RbcL-GFP strain, respectively). Primers used are described in Table 2.

TABLE 2 Primers Primer ID Purpose Sequence pUC19 CcmM-N GGTGCACTACTAGTACAATCTGC speI fwd deletion (SEQ ID NO: 39) pUC19 CcmM-N GTGAAATACCGCACTAGTGCGTAAG speI rv deletion (SEQ ID NO: 40) FR left CcmM-N CTTTCATCTTGAATTCCGACTCTTTAGG (ccmL-O) fwd deletion (SEQ ID NO: 41) FR left CcmM-N GCTCGGCATATGCTAACCTC (ccmL-O) rv deletion (SEQ ID NO: 42) FR right CcmM-N GGGAGGTTAGCATATGCTCTAGAAGCTGCAGG (ccmL-O) fwd deletion (SEQ ID NO: 43) FR right CcmM-N CTACTGAGTCCGAAGCTTTCAGC (ccmL-O) rv deletion (SEQ ID NO: 44) Km^(R)/SacB CcmM-N GAATTATAACCATATGCATCCTAGG fwd deletion (SEQ ID NO: 45) Km^(R)/SacB CcmM-N TCCCGTCTAGACAGCGTAATG rv deletion (SEQ ID NO: 46) CcaA ndeI ccmC, GAGTATCACTCATATGCGCAAGC fwd ccaA-M35 (SEQ ID NO: 47) CcaA BamHI ccaA-M35 CTTCGGGATCCGCTAGCATTG rv (SEQ ID NO: 48) SSLDs-CcmN ccaA-M35 TAGCGAGGCAAGATCTGTGAGC bglII fwd- (SEQ ID NO: 49) SSLDs-CcmN ccaA-M35 CCTGCAGCTTCTAGAGCTGCTGTG xhoI rv- (SEQ ID NO: 50) CcaA_((short)) ccmC GTTGTTGTTCGGATCCCAACCAAC bamHI rv (SEQ ID NO: 51) EP bglII ccmC, CCCAGATCTGGAGGCAGTGTCTACGGCAAGGAAC fwd M35-EP (SEQ ID NO: 52) EP NcoI ccmC, CGTGGCCATGGCTTCTTGGGAGAGC rv M35-EP (SEQ ID NO: 53) ccaA_((short)) ccmC GCCCTTGTCAGATCTCGCAAGCTCATCG bglII fwd (SEQ ID NO: 54) SSLDs ccmC, CTAGCGAGCATATGACCGTGAGCGC ndeI fwd M35-EP (SEQ ID NO: 55) SSLDs ccmC, CAGGATCCTCCCGGCTTTTGTTAGAGC bamHI rv M35-EP (SEQ ID NO: 56) FR left CcaA CAGCGGCCGCGCCTAGTGC (ccaA) notI fwd deletion (SEQ ID NO: 57) FR left CcaA GCTTGCGCATCTCGAGTGATACTCGGGAC (ccaA) xhoI rv deletion (SEQ ID NO: 58) FR right CcaA GCGGCAATTCTAGATAGGATCGAAGCATC (ccaA) xbaI fwd deletion (SEQ ID NO: 59) FR right CcaA TACCCATGGACTCAAGCGCTCATTGCCAG (ccaA) ncoI rv deletion (SEQ ID NO: 60) Gm^(R) xhoI CcaA GGTACCGAGCTCGAGTTGACATAAGC fwd deletion (SEQ ID NO: 61) Gm^(R) xbaI CcaA TCCGCGGCTCTAGAGCCGATC rv deletion (SEQ ID NO: 62) Primer A Screening TGCCTATTGCGGTTGGAATG (SEQ ID NO: 63) Primer B Screening AATCATGATGCACGCCCTTG (SEQ ID NO: 64) Primer C Screening AATCATGATGCACGCCCTTG (SEQ ID NO: 65) Primer D Screening TTAGCCGATTTGAGCATGGC (SEQ ID NO: 66) Primer E Screening CAGCTTTGAACATTGAGCATGTTGTG (SEQ ID NO: 67) Primer F Screening ATTGCCGCGATAAATCCGCTG (SEQ ID NO: 68) Structural Modeling

The predicted domains obtained (FIG. 1A) were used as input for the automated mode of the SwissModel (Biasini et al., 2014) server. The EP was manually added to the predicted structure of CcmC using the software Chimera (Pettersen et al., 2004).

Spectrophotometric Measurements

Culture growth was monitored as the change in optical density at 730 nm (OD₇₃₀). Chl α concentration was determined by absorbance measurements (at 663 nm) of methanol extracts from 1-ml culture aliquots and calculated according to Lichtenthaler (Lichtenthaler, 1987). Total cell spectra were obtained from 1-ml aliquots of cultures in exponential growth phase, which were diluted to OD₇₃₀=0.3, and the obtained spectra were normalized to that of Chl α (OD₆₆₃). Doubling times were calculated using the exponential regression curve fitting online tool available at website doubling-time.com/compute.php. All measurements were performed at least in triplicate from aliquots from different cultures (using the same inoculum from a BG-11 agar plate). All measurements were performed in a Nanodrop2000C spectrophotometer (Thermo Scientific, USA).

PCR and Immunoblot Analysis

Standard PCR was performed as described in the manufacturer's protocol using EconoTaq Plus Green 2X (Lucigen, USA) and gene-specific primer pairs (Table 2). For protein extraction, pellets from 50 ml culture aliquots were resuspended in 1 ml of lysis buffer (25 mM HEPES-NaOH pH 7, 15 mM CaCl₂, 5 mM MgCl₂, 15% Glycerol, 200 μM PMSF and cOmplete, Mini protease inhibitor (Roche)) and broken in a BeadBug homogenizer (Biospec Products, USA), by beating for 6 cycles of 30 seconds and 2 minutes of incubation in ice between each cycle. After 20 minutes of centrifugation at 20000 rcf, 15-μl aliquots plus SDS loading dye were loaded onto an acrylamide gel (without boiling the sample) for SDS-PAGE. SDS-PAGE and immunoblot analysis were performed according to the manufacturer's protocol (BioRad's bulletin 6376) using a polyclonal antibody from rabbit against Syn 7942 CcmM (dilution 1:5000) (Rothamstead Research, UK) as a primary antibody and Goat Anti-Rabbit IgG-HRP (Dilution 1:7000) (Life Tech. #656120) as secondary antibody and 1-Step Ultra TMB-Blotting Solution as substrate (Thermo #37574). For densitometries, total protein extract samples from three independent cultures were normalized according to the peak absorbance at 663 nm, loaded at four decreasing serial dilutions, and blotted as described using Anti-RbcL antibody (Agrisera Cat. AS03 037) at a dilution of 1:10000. Densitometry measurements were performed on the different immunoblots using ImageJ software (Schneider et al., 2012).

Oxygen Evolution

Two-ml aliquots were harvested from exponential-phase cultures, supplemented with 10 mM bicarbonate prior to the measurement, and the steady-state rate of oxygen evolution was determined at saturating light intensity (950 moles photons m⁻²s⁻¹) and 30° C. using an LMI-6000 illuminator (Dolan-Jenner, USA) and an Oxygraph Plus Clark-type electrode (Hansatech, UK).

Fluorescence and Electron Microscopy

Cultures grown to OD₇₃₀=0.5 in 3% CO₂ were transferred to air and grown overnight. For fluorescence microscopy, 1-ml aliquots were concentrated by centrifugation (1500 rcf for 5 minutes and resuspended in 100 μl of BG11) and visualized (autofluorescence and GFP) using a Zeiss Axio Observer.D1 inverted microscope. For electron microscopy, pellets from 50-ml aliquots were chemically fixed with 2% glutaraldehyde in 50 mM phosphate buffer for 2 hours at room temperature, followed by 1% osmium tetroxide for 2 hours at room temperature, and block stained with 2% aqueous uranyl acetate overnight at 4° C. Cells were dehydrated in an increasing acetone series (2 minutes at 37° C.; 20% acetone increments) and embedded in Spurr's resin (15 minutes at 37° C.; 25% increments) using an MS-9000 Laboratory Microwave Oven (Electron Microscopy Science, USA). Sections (70 nm thick) were cut on a MYX ultramicrotome (RMC Products, USA), positively stained with 6% uranyl acetate and Reynolds lead citrate (Reynolds, 1963) and visualized on a JEM 100CX II transmission electron microscope (JEOL) equipped with an Orius SC200-830 CCD camera (Gatan Inc., USA).

Quantum Efficiency of Photosystem II

F_(v)/F_(m) was determined in triplicate using 4-ml culture aliquots from biological replicates at exponential phase in cells dark adapted for three minutes as described previously (Cameron et al., 2013). Briefly, aliquots were diluted with BG-11 immediately before dark adaptation to a chlorophyll concentration of ˜1-2 μg/ml and measured using an Aquapen AP100 (Photon Systems Instruments, Czech Republic). Measurement started at time=0 h when the cultures were transferred from 3% CO₂ to air.

Sequences

Sequences can be found in the GenBank/EMBL data libraries. For example, an amino acid sequence for a Synechococcus elongatus PCC 7942 carbonate dehydratase (CcmM: Synpcc7942_1423) is available as accession number ABB57453 (SEQ ID NO:69).

1 MPSPTTVPVA TAGRLAEPYI DPAAQVHAIA SIIGDVRIAA 41 GVRVAAGVSI RADEGAPFQV GKESILQEGA VIHGLEYGRV 81 LGDDQADYSV WIGQRVAITH KALIHGPAYL GDDCFVGFRS 121 TVFNARVGAG SVIMMHALVQ DVEIPPGRYV PSGAIITTQQ 161 QADRLPEVRP EDREFARHII GSPPVIVRST PAATADFHST 201 PTPSPLRPSS SEATTVSAYN GQGRLSSEVI TQVRSLLNQG 241 YRIGTEHADK RRFRTSSWQP CAPIQSTNER QVLSELENCL 281 SEHEGEYVRL LGIDTNTRSR VFEALIQRPD GSVPESLGSQ 321 PVAVASGGGR QSSYASVSGN LSAEVVNKVR NLLAQGYRIG 361 TEHADKRRFR TSSWQSCAPI QSSNERQVLA ELENCLSEHE 401 GEYVRLLGID TASRSRVFEA LIQDPQGPVG SAKAAAAPVS 441 SATPSSHSYT SNGSSSSDVA GQVRGLLAQG YRISAEVADK 481 RRFQTSSWQS LPALSGQSEA TVLPALESIL QEHKGKYVRL 521 IGIDPAARRR VAELLIQKP

An amino acid sequence for a Synechococcus elongatus PCC 7942 carbon dioxide concentrating mechanism protein (CcmN: Synpcc7942_1424) is available as accession number ABB57454 (SEQ ID NO:70).

1 MHLPPLEPPI SDRYFASGEV TIAADVVIAP GVLLIAEADS 41 RIEIASGVCI GLGSVIHARG GAIIIQAGAL LAAGVLIVGQ 81 SIVGRQACLG ASTTLVNTSI EAGGVTAPGS LLSAETPPTT 121 ATVSSSEPAG RSPQSSAIAH PTKVYGKEQF LRMRQSMFPD 161 R

An amino acid sequence for a Synechococcus elongatus PCC 7942 Carbonate dehydratase (CcaA; Synpcc7942_1447) is available as accession number ABB57477.1 (SEQ ID NO:71).

1 MRKLIEGLRH FRTSYYPSHR DLFEQFAKGQ HPRVLFITCS 41 DSRIDPNLIT QSGMGELFVI RNAGNLIPPF GAANGGEGAS 81 IEYAIAALNI EHVVVCGHSH CGAMKGLLKL NQLQEDMPLV 121 YDWLQHAQAT RRLVLDNYSG YETDDLVEIL VAENVLTQIE 161 NLKTYPIVRS RLFQGKLQIF GWIYEVESGE VLQISRTSSD 201 DTGIDECPVR LPGSQEKAIL GRCVVPLTEE VAVAPPEPEP 241 VIAAVAAPPA NYSSRGWLAP EQQQRIYRGN AS

Example 2: Design of a Chimeric Protein that Supports Native Core Assembly and Cell Growth in Air

This Example describes construction of chimeric proteins that assemble into a carboxysome core.

The design took into consideration observations that proteins evolve via domain fusions that are reflective of protein-protein interactions. The inventors predicted the domain boundaries in the CcmM, CcmN and CcaA proteins from Synechococcus elongatus PCC 7942 (FIG. 1A) using InterPro (Hunter et al., 2012). Three chimeric genes were then constructed encoding proteins that could assemble into a carboxysome core:

1) a ccaA-M35 fusion construct, where the γ-CA domain (Pfam00132) of CcmM was replaced by β-CA (Pfam00484) (FIG. 1D);

2) a M35-EP fusion construct, where three SSLD domains (Pfam00101) and their native linkers were fused to the EP (FIG. 1E); and

3) M35-ccaA_((short))-EP fusion construct, containing three SSLDs and their native linkers, the β-CA, CcaA with a short segment of its C-terminal tail as a linker, and the EP from the C-terminus of CcmN (FIGS. 1B, 1F-1H).

A gene coding for a green fluorescent protein (GFP)-labeled large subunit of Rubisco (rbcL-GFP) was inserted into each strain for in vivo visualization of carboxysome formation by fluorescence microscopy (Savage et al., 2010). To test whether the chimeric proteins can assemble into a carboxysome core, the Synechococcus elongatus PCC 7942 ccmM and ccmN were replaced with selectable marker genes (COREΔ2/RbcL-GFP strain; hcr phenotype). The chimeric genes were then transformed via double homologous recombination to replace the selectable markers of the COREΔ2/RbcL-GFP strain (placing the genes under the same regulation of the ccm operon genes) using growth in air for positive selection. In the case of ccaA-M35, the ccmN gene was reintroduced in the same vector.

Only M35-ccaA_((short))-EP expression was able to rescue the hcr phenotype. This construct was named CcmC where the final “C” was for chimeric (FIG. 1H). The resulting strain (COREΔ2/CcmC/RbcL-GFP) contained the original ccaA in its genome. Therefore, to further substantiate the evident functional rescue by CcmC, the native ccaA was replaced with a gentamycin resistance gene (resulting in strain COREΔ3/CcmC/RbcL-GFP). This triple mutant strain was able to grow in air.

The presence or absence of ccmM, ccmN and ccaA was confirmed by PCR. Sequencing of the region between ccmL and ccmO further indicated that ccmC was integrated into the ccm operon. The CCM insertion site sequence is shown below (SEQ ID NO:72), where the ccmC DNA insert is identified in bold and with underlining, and the portion of the genomic ccmK2 gene disrupted by the ccmC DNA insert is shown in bold (at the beginning of the SEQ ID NO:72 sequence).

1 AGCCGCGGCA GTCAAGCGCG CCATGTGCGC GATTGTCAGG 41 AACGACCGGT TGATGCAGCT GTCATTGCCA TCATCGATAC 81 GGTCAACGTG GAAAACCGCT CCGTCTACGA CAAACGCGAG 121 CACAGCTAAT GGGCAGGGAT TGAATCCCTG CTGGTCATTG 161 ATCTGGATTG AGCCCAGGCT TGGGAGGTTA GCAT ATGACC 201 GTGAGCGCTT ATAACGGCCA AGGCCGACTC AGTTCCGAAG 241 TCATCACCCA AGTCCGGAGT TTGCTGAACC AGGGCTATCG 281 GATTGGGACG GAACATGCGG ACAAGCGCCG CTTCCGGACT 321 AGCTCTTGGC AGCCCTGCGC GCCGATTCAA AGCACGAACG 361 AGCGCCAGGT CTTGAGCGAA CTGGAAAATT GTCTGAGCGA 401 ACACGAAGGT GAATACGTTC GCTTGCTCGG CATCGATACC 441 AATACTCGCA GCCGTGTTTT TGAAGCCCTG ATTCAACGGC 481 CCGATGGTTC GGTTCCTGAA TCGCTGGGGA GCCAACCGGT 521 GGCAGTCGCT TCCGGTGGTG GCCGTCAGAG CAGCTATGCC 561 AGCGTCAGCG GCAACCTCTC AGCAGAAGTG GTCAATAAAG 601 TCCGCAACCT CTTAGCCCAA GGCTATCGGA TTGGGACGGA 641 ACATGCAGAC AAGCGCCGCT TTCGGACTAG CTCTTGGCAG 681 TCCTGCGCAC CGATTCAAAG TTCGAATGAG CGCCAGGTTC 721 TGGCTGAACT GGAAAACTGT CTGAGCGAGC ACGAAGGTGA 761 GTACGTTCGC CTGCTGGGCA TCGACACTGC TAGCCGCAGT 801 CGTGTTTTTG AAGCCCTGAT CCAAGATCCC CAAGGACCGG 841 TGGGTTCCGC CAAAGCGGCC GCCGCACCTG TGAGTTCGGC 881 AACGCCCAGC AGCCACAGCT ACACCTCAAA TGGATCGAGT 921 TCGAGCGATG TCGCTGGACA GGTTCGGGGT CTGCTAGCCC 961 AAGGCTACCG GATCAGTGCG GAAGTCGCCG ATAAGCGTCG 1001 CTTCCAAACC AGCTCTTGGC AGAGTTTGCC GGCTCTGAGT 1041 GGCCAGAGCG AAGCAACTGT CTTGCCTGCT TTGGAGTCAA 1081 TTCTGCAAGA GCACAAGGGT AAGTATGTGC GCCTGATTGG 1121 GATTGACCCT GCGGCTCGTC GTCGCGTGGC TGAACTGTTG 1161 ATTCAAAAGC CGGGATCTCG CAAGCTCATC GAGGGGTTAC 1201 GGCATTTCCG TACGTCCTAC TACCCGTCTC ATCGGGACCT 1241 GTTCGAGCAG TTTGCCAAAG GTCAGCACCC TCGAGTCCTG 1281 TTCATTACCT GCTCAGACTC GCGCATTGAC CCTAACCTCA 1321 TTACCCAGTC GGGCATGGGT GAGCTGTTCG TCATTCGCAA 1361 CGCTGGCAAT CTGATCCCGC CCTTCGGTGC CGCCAACGGT 1401 GGTGAAGGGG CATCGATCGA ATACGCGATC GCAGCTTTGA 1441 ACATTGAGCA TGTTGTGGTC TGCGGTCACT CGCACTGCGG 1481 TGCGATGAAA GGGCTGCTCA AGCTCAATCA GCTGCAAGAG 1521 GACATGCCGC TGGTCTATGA CTGGCTGCAG CATGCCCAAG 1561 CCACCCGCCG CCTAGTCTTG GATAACTACA GCGGTTATGA 1601 GACTGACGAC TTGGTAGAGA TTCTGGTCGC CGAGAATGTG 1641 CTGACGCAGA TCGAGAACCT TAAGACCTAC CCGATCGTGC 1681 GATCGCGCCT TTTCCAAGGC AAGCTGCAGA TTTTTGGCTG 1721 GATTTATGAA GTTGAAAGCG GCGAGGTCTT GCAGATTAGC 1761 CGTACCAGCA GTGATGACAC AGGCATTGAT GAATGTCCAG 1801 TGCGTTTGCC CGGCAGCCAG GAGAAAGCCA TTCTCGGTCG 1841 TTGTGTCGTC CCCCTGACCG AAGAAGTGGC CGTTGCTCCA 1881 CCAGAGCCGG AGCCTGTGAT CGCGGCTGTG GCGGCTCCAC 1921 CCGCCAACTA CTCCAGTCGC GGTTGGTTGG GATCTGGAGG 1961 CAGTGTCTAC GGCAAGGAAC AGTTTTTGCG GATGCGCCAG 2001 AGCATGTTCC CCGATCGCTA A GATGTGCAC AGCAGCTCTA 2041 GGAGCTGCAG GGTACT

The portion of the sequence of the ccmL gene at the ccmC integration site is shown below (SEQ ID NO:73).

1 AGCCGCGGCA GTCAAGCGCG CCATGTGCGC GATTGTCAGG 41 AACGACCGGT TGATGCAGCT GTCATTGCCA TCATCGATAC 81 GGTCAACGTG GAAAACCGCT CCGTCTACGA CAAACGCGAG 121 CACAGCTAA

Protein screening by immunoblot using polyclonal anti-CcmM antibodies showed no cross-reactivity with a total protein extract of the COREΔ2/RbcL-GFP strain, confirming the absence of those proteins (FIG. 2). In contrast, signals at ˜37 kDa (major) and at ˜63 kDa (minor) were observed in wild type and in the control strain expressing rbcL-GFP (hereafter Wild-type/RbcL-GFP strain), corresponding to the two forms of CcmM required for carboxysome assembly in Wild-type Syn 7942 carboxysomes (corresponding to M35 and full-length CcmM (So et al., 2002b; Long et al., 2010). These two bands are absent in the COREΔ2/CcmC/RbcL-GFP and COREΔ3/CcmC/RbcL-GFP strains, and replaced by cross-reactivity at ˜75 kDa, corresponding to the fusion protein (predicted mass of 67 kDa; FIG. 2).

Example 3: CcmC Replaces Four Proteins of the β-carboxysome Core

This Example illustrates assembly of CcmC into functioning carboxysomes.

Fluorescence and transmission electron microscopy were used to assay for formation of carboxysomes (FIG. 3). In the wild-type/RbcL-GFP strain, the carboxysomes were in the typical arrangement, along the longitudinal axes of the cells (FIG. 3 panel A). RbcL-GFP in the COREΔ2/RbcL-GFP strain was diffuse throughout the cell, as expected for strains lacking carboxysomes (Cameron et al., 2013) (FIG. 3 panel B). Occasional polar foci (n=150/556) were observed. Such polar foci may be due to misfolded and aggregated labeled protein. For example, polar localization of protein aggregates (Rokney et al., 2009) and false foci (Landgraf et al., 2012) have been observed in E. coli. Such foci may also be due to interaction with the remaining gene products of the carboxysome operon. They are not indicative of carboxysome formation, as the COREΔ2/RbcL-GFP strain has an her phenotype (i.e., a high CO₂-requiring phenotype).

In contrast, abundant GFP-labeled carboxysomes were observed in the mutant strains COREΔ2/CcmC/RbcL-GFP and COREΔ3/CcmC/RbcL-GFP (FIG. 3 panels C and D, respectively). Although occasionally clustered, the carboxysomes still localized along the longitudinal axis of the cell (FIG. 3 panels C and D, respectively).

The average carboxysome number (fluorescent puncta across the longitudinal plane) per cell in the wild-type/RbcL-GFP strain was 3.7±1.1 (FIG. 4A). The average carboxysome number was somewhat higher in the COREΔ2/CcmC/RbcL-GFP strain (average 6.4±1.8) and in the COREΔ3/CcmC/RbcL-GFP strain (average 6.4±2.0) (FIG. 4A).

The amount of Rubisco protein per mg Chlorophyll a (Chl α) protein in the different strains was compared by immunoblotting using antibodies against the large subunit RbcL. Both COREΔ2/CcmC/RbcL-GFP and COREΔ3/CcmC/RbcL-GFP strains contained more than a 2-fold increase in RbcL relative to the Wild-type/RbcL strain (FIG. 4B).

Analysis by transmission electron microscopy further confirmed carboxysome formation of native (FIG. 3 panel E) and streamlined carboxysomes (FIG. 3 panels G and H).

The chimeric carboxysomes were smaller than wild type carboxysomes. As illustrated in FIG. 4C, the average carboxysome diameter for wild-type/RbcL-GFP carboxysomes was 185±28 nm, but the average diameter of COREΔ2/CcmC carboxysomes was 103±25 nm and the average diameter of COREΔ3/CcmC carboxysomes was 95±19 nm. In addition, the CcmC strains typically had more carboxysomes and the CcmC carboxysomes tended to be more clustered compared to the wild-type carboxysomes.

Abnormally shaped carboxysomes were occasionally observed (“rod carboxysomes”) in the CcmC strains but these have also been observed in wild type cyanobacteria (Gantt and Conti, 1969). Researchers have proposed that such rod carboxysomes may be a type of intermediate during carboxysome formation (Chen et al., 2013). Based on studies by the inventors, these rod carboxysomes could also be indicative of a deficiency in CA activity, as carboxysome aggregation and morphological variation were observed in the control strain ΔCcaA/RbcL-GFP (data not shown).

To determine if the reengineered carboxysomes function comparably to the Wild-type/RbcL-GFP carboxysomes, the growth of cells was analyzed at the exponential growth phase under high CO₂ (5%) and low CO₂ (air) conditions. No growth difference was observed between the strains when incubated in high CO₂ (FIG. 5B), because under these conditions, cyanobacterial CO₂ fixation does not depend upon proper carboxysome formation. As expected, the COREΔ2/RbcL-GFP strain (without the CcmC construct) failed to grow in air, whereas in air the other strains were able to grow (FIG. 5A). The COREΔ2/CcmC/RbcL-GFP strain had the fastest doubling time among the reengineered strains tested, while the growth rates of COREΔ3/CcmC/RbcL-GFP and the Wild-type/RbcL-GFP strain are comparable (FIG. 5A-5B).

Example 4: Physiology of a Cyanobacterial Strain with a Streamlined Carboxysome

This Example illustrates some of the physiological characteristics of a triple deletion strain containing carboxysomes with synthetic cores (COREΔ3/CcmC/RbcL-GFP).

The COREΔ3/CcmC/RbcL-GFP strain has pigmentation differences when compared to Wild-type/RbcL-GFP (FIG. 6A). Such a difference could be attributed to decreased phycobilisome content.

The relative photosynthetic capacities of photosystem II were measure through quantification of chlorophyll fluorescence in dark adapted cells (F_(v)/F_(m)) upon transfer of the cultures from 3% CO₂ to air (FIG. 6B) The F_(v)/F_(m) is widely used as a measure of the efficiency of the photosynthetic electron transport chain, which generates the ATP and reducing power that is consumed by the Calvin-Benson-Bassham (CBB) cycle (Baker, 2008). Accordingly, F_(v)/F_(m) has been used as a proxy for carboxysome function. For example, carboxysome-deficient strains of Syn 7942 have an F_(v)/F_(m) approximating zero in 3% CO₂ (Cameron et al., 2013).

As illustrated in FIG. 6B, while the F_(v)/F_(m) of Wild-type/RbcL-GFP remains relatively constant (solid line), a sharp decrease in F_(v)/F_(m) relative to the high-CO₂ values is observed in both mutant core strains. The F_(v)/F_(m) in the COREΔ2/RbcL-GFP control strain declined towards zero and did not recover (dashed, dotted line in FIG. 6B). However, the COREΔ3/CcmC/RbcL-GFP strain (dashed line in FIG. 6B) adapted within about 10 hr after the CO₂ step-down and eventually reached the same fluorescence levels as the wild-type/RbcL-GFP strain.

As an additional, complementary measure of photosynthetic activity, the oxygen evolution rates of air-grown cultures were compared at high light intensity (950 μmoles photons m⁻² s⁻¹). As shown in FIG. 6C, the wild-type/RbcL-GFP cells (dark gray bar) produced more oxygen than the COREΔ3/CcmC/RbcL-GFP strain (light gray bar) The amounts of oxygen produced by the wild-type/RbcL-GFP cells was 2.9±1.0 μmoles O₂ μg Chl_(a) ⁻¹ min⁻¹ compared to 1.3±0.5 μmoles O₂ μg Chl_(a) ⁻¹ min⁻¹ for the COREΔ3/CcmC/RbcL-GFP.

These results indicate that the altered composition of the core has a net effect on the physiology of the cell relative to the Wild-type/RbcL-GFP control. Nevertheless, the reengineered core is immediately able to effectively support functional carboxysome assembly (FIG. 3 panels C and D) and photosynthesis (FIG. 5A-5B).

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All patents and publications referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced patent or publication is hereby specifically incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such cited patents or publications.

The following statements are intended to describe and summarize various embodiments of the invention according to the foregoing description in the specification.

Statements:

-   -   1. A fusion protein comprising at least two small subunit-like         domains (SSLDs), at least one carbonic anhydrase (CA) domain,         and at least one encapsulation peptide (EP).     -   2. The fusion protein of statement 1, wherein the at least one         carbonic anhydrase (CA) domain is flanked on one side by the at         least two small subunit-like domains (SSLDs), and by the at         least one encapsulation peptide (EP) on the other side.     -   3. The fusion protein of statement 1 or 2, wherein the at least         two small subunit-like domains (SSLDs) comprise scaffolding         domains that can bind or nucleate with ribulose-1,5-bisphosphate         carboxylase/oxygenase (Rubisco).     -   4. The fusion protein of statement 1, 2, or 3, wherein the at         least two small subunit-like domains (SSLDs) can nucleate with         ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), and         the Rubisco can synthesize 3-phosphoglycerate (3-PGA).     -   5. The fusion protein of statement 1-3, or 4, wherein the at         least two small subunit-like domains (SSLDs) comprise at least         40% sequence identity, or at least 50% sequence identity, or at         least 60% sequence identity, or at least 70% sequence identity,         or at least 80% sequence identity, or at least 90% sequence         identity, or at least 95% sequence identity, or at least 96%         sequence identity, or at least 97% sequence identity, or at         least 98% sequence identity, or at least 99% sequence identity,         or 60-99% sequence identity, or 70-99% sequence identity, or         80-99% sequence identity, or 90-95% sequence identity, or 90-99%         sequence identity, or 95-97% sequence identity, or 97-99%         sequence identity, or 100% sequence identity (or         complementarity) with any of SEQ ID NOs:1-11, 37, 75, 76, or 77.     -   6. The fusion protein of statement 1-4 or 5, wherein the at         least two small subunit-like domains (SSLDs) comprise at least         40% sequence identity, or at least 50% sequence identity, or at         least 60% sequence identity, or at least 70% sequence identity,         or at least 80% sequence identity, or at least 90% sequence         identity, or at least 95% sequence identity, or at least 96%         sequence identity, or at least 97% sequence identity, or at         least 98% sequence identity, or at least 99% sequence identity,         or 60-99% sequence identity, or 70-99% sequence identity, or         80-99% sequence identity, or 90-95% sequence identity, or 90-99%         sequence identity, or 95-97% sequence identity, or 97-99%         sequence identity, or 100% sequence identity (or         complementarity) with any of SEQ ID NOs:5-11, 37, 75, 76, or 77.     -   7. The fusion protein of statement 1-5 or 6, wherein the at         least one carbonic anhydrase domain converts bicarbonate to         carbon dioxide.     -   8. The fusion protein of statement 1-6 or 7, wherein the at         least one carbonic anhydrase domain comprises at least 40%         sequence identity, or at least 50% sequence identity, or at         least 60% sequence identity, or at least 70% sequence identity,         or at least 80% sequence identity, or at least 90% sequence         identity, or at least 95% sequence identity, or at least 96%         sequence identity, or at least 97% sequence identity, or at         least 98% sequence identity, or at least 99% sequence identity,         or 60-99% sequence identity, or 70-99% sequence identity, or         80-99% sequence identity, or 90-95% sequence identity, or 90-99%         sequence identity, or 95-97% sequence identity, or 97-99%         sequence identity, or 100% sequence identity with any of SEQ ID         NOs:17-21 or 71.     -   9. The fusion protein of statement 1-7 or 8, wherein the at         least one encapsulation peptide interacts with and/or binds one         or more carboxysome shell protein.     -   10. The fusion protein of statement 1-8 or 9, wherein the at         least one encapsulation peptide comprises at least 40% sequence         identity, or at least 50% sequence identity, or at least 60%         sequence identity, or at least 70% sequence identity, or at         least 80% sequence identity, or at least 90% sequence identity,         or at least 95% sequence identity, or at least 96% sequence         identity, or at least 97% sequence identity, or at least 98%         sequence identity, or at least 99% sequence identity, or 60-99%         sequence identity, or 70-99% sequence identity, or 80-99%         sequence identity, or 90-95% sequence identity, or 90-99%         sequence identity, or 95-97% sequence identity, or 97-99%         sequence identity, or 100% sequence identity (or         complementarity) with any of SEQ ID NOs:12-16.     -   11. An expression cassette comprising a promoter operably linked         to a nucleic acid segment encoding the fusion protein of         statement 1-9 or 10.     -   12. The expression cassette of statement 11, wherein the         promoter is a constitutive promoter, inducible promoter,         regulated promoter, cell specific promoter, or synthetic         promoter.     -   13. The expression cassette of statement 11 or 12, wherein the         promoter is active before or during log phase growth of cells         comprising the expression cassette.     -   14. The expression cassette of statement 11, 12, or 13, wherein         the promoter is active after log phase growth of cells         comprising the expression cassette.     -   15. The expression cassette of statement 11-13 or 14, wherein         the nucleic acid segment encoding the fusion protein comprises         at least 40% sequence identity, or at least 50% sequence         identity, or at least 60% sequence identity, or at least 70%         sequence identity, or at least 80% sequence identity, or at         least 90% sequence identity, or at least 95% sequence identity,         or at least 96% sequence identity, or at least 97% sequence         identity, or at least 98% sequence identity, or at least 99%         sequence identity, or 60-99% sequence identity, or 70-99%         sequence identity, or 80-99% sequence identity, or 90-95%         sequence identity, or 90-99% sequence identity, or 95-97%         sequence identity, or 97-99% sequence identity, or 100% sequence         identity (or complementarity) with any of SEQ ID NOs:26-29.     -   16. A cell comprising the expression cassette of statement 11-14         or 15.     -   17. An expression vector comprising the expression cassette of         statement 11-14 or 15.     -   18. A cell comprising the expression vector of statement 16.     -   19. An organism comprising the fusion protein of statement 1-9         or 10.     -   20. An organism comprising a nucleic acid segment encoding the         fusion protein of statement 1-9 or 10.     -   21. An organism comprising the expression cassette of statement         11-15, or the expression vector of statement 17.     -   22. A method comprising expressing the fusion protein of         statement 1-9 or 10 in a cell.     -   23. A method comprising expressing the fusion protein of         statement 1-9 or 10 from a nucleic acid in a cell.     -   24. A method comprising expressing the fusion protein of         statement 1-9 or 10 from a heterologous nucleic acid in a cell.     -   25. A method comprising expressing a fusion protein encoded by         the expression cassette of statement 11-14 or 15 in a cell.     -   26. A method for carbon fixation comprising expressing the         fusion protein of statement 1-9 or 10 in a cell.     -   27. The method of statement 26, wherein the cell is a         cyanobacteria, a bacteria, a plant cell, or an algae (e.g., a         microalgae).     -   28. A method for carbon fixation comprising culturing a cell         comprising the expression cassette of statement 11-14 or 15.     -   29. The method of statement 28, wherein the cell is a         cyanobacteria, a bacteria, a plant cell, or an algae (e.g., a         microalgae).     -   30. A method for oxygen evolution comprising culturing a cell         comprising the expression cassette of statement 11-14 or 15.     -   31. A method comprising culturing a cell that can express the         fusion protein of statement 1-9 or 10, and that can synthesize a         product selected from a carbohydrate, sugar, protein, fatty         acid, oil, biomass, alcohol, isobutyraldehyde, butanol, ethanol,         propanediol, or isoprene.     -   32. The method of statement 31, wherein the cell is a         cyanobacteria, a bacteria, a plant cell, or an algae (e.g., a         microalgae).

The specific compositions and methods described herein are representative, exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims and statements of the invention.

The invention illustratively described herein may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. The methods and processes illustratively described herein may be practiced in differing orders of steps, and the methods and processes are not necessarily restricted to the orders of steps indicated herein or in the claims.

As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a plant” or “a seed” or “a cell” includes a plurality of such plants, seeds or cells, and so forth. In this document, the term “or” is used to refer to a nonexclusive or, such that “A or B” includes “A but not B,” “B but not A,” and “A and B,” unless otherwise indicated.

Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants.

The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

The Abstract is provided to comply with 37 C.F.R. § 1.72(b) to allow the reader to quickly ascertain the nature and gist of the technical disclosure. The Abstract is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. 

What is claimed:
 1. A fusion protein comprising a polypeptide comprising at least two fused small subunit-like domains (SSLDs) that can bind or nucleate with ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), each SSLD comprising at least 95% sequence identity to any of SEQ ID NO: 75, 76 or 77, one SSLD fused to at least one domain having carbonic anhydrase activity, and at least one encapsulation peptide that interacts or binds with to one or more carboxysome shell protein, each encapsulation peptide comprising at least 95% sequence identity to any of SEQ ID NO: 14, 15 or 16 fused to the at least one carbonic anhydrase domain.
 2. The fusion protein of claim 1, wherein the Rubisco can synthesize 3-phosphoglycerate (3-PGA).
 3. The fusion protein of claim 1, wherein the at least one carbonic anhydrase domain comprises at least 95% sequence identity to any of SEQ ID NO:17-21 or
 71. 